Application of Low-Molecular-Weight Hyaluronic Acid (LMW-HA) Fragments

ABSTRACT

Various pharmaceutical applications of low-molecular-weight hyaluronic acid (LMW-HA) fragments include: treating tu-mors, conjunctival diseases, xerophthalmia, vitreous opacity, myofascitis, arthritis, cardiovascular diseases, cerebral infarction, dysmenorrhea, endometriosis, periodontal diseases, herpes zoster, burns, pains, pruritus, acute pancreatitis, and postoperative abdominal mucosal adhesions, helping with body recovery after chemotherapy and facial cosmesis, reducing subcutaneous fat etc. Moreover, an injection containing the LMW-HA fragments and a preparation method thereof are disclosed. The injection is injected into the subcutaneous fat layer of the abdomen for facial cosmesis and anti-aging.

CROSS REFERENCE TO THE RELATED APPLICATIONS

This application is a divisional application (DIV) of non-provisionalapplication Ser. No. 16/754,756 filed on Apr. 9, 2020, which is thenational phase entry of International Application No. PCT/CN2017/081796,filed on Apr. 25, 2017, which is based upon and claims priority toChinese Patent Application No. 201610264221.9, filed on Apr. 25, 2016,and Chinese Patent Application No. 201710272309.X, filed on Apr. 24,2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to the application field of low-molecular-weighthyaluronic acid (LMW-HA) fragments, in particular to an application ofLMW-HA fragments in preparing drugs for treating a variety of diseasesand morbid conditions including solid tumors, as well as the usage infacial cosmetology and anti-aging, enhancing physical strength andenergy, reducing subcutaneous fat, etc.

BACKGROUND

Hyaluronic acid (HA), also known as hyaluronan, is a long-chainpolysaccharide consisted of D-glucuronic acid and N-acetylglucosamine,with the disaccharide unit up to 25 KD. Tissues such as humansubcutaneous tissue, epidermis, oropharyngeal mucosa, etc. contain alarge amount of high-molecular-weight hyaluronic acid (HMW-HA). In otherwords, HMW-HA is the basic construction material for human skin, mucousmembranes, subcutaneous tissues, etc., and it has the functions of waterretention and moisturizing. The existing HA products in the market aremainly the products with HMW-HA as the main functional ingredient forfacial care or facial cosmetology.

In recent years, a series of studies have suggested that LMW-HA andHMW-HA, which are distinguished by molecular weight, demonstratefunctional differences in small animal experiments and cell levelstudies. The mainstream references have indicated that LMW-HA is adegraded byproduct of inflamed tissues and is co-located with thedistribution of inflammation, and it has the function of inducing andpromoting inflammatory responses. The main references include:

-   [1]. Jaime M. Cyphert, Carol S. Trempus, and Stavros Garantziotis,    Size Matters: Molecular Weight Specificity of Hyaluronan Effects in    Cell Biology (Review Article), International Journal of Cell    Biology, Volume 2015 (2015), Article ID 563818.-   [2]. Jiang D1, Liang J, Noble P W. Hyaluronan as an immune regulator    in human diseases. Physiol Rev. 2011 January; 91(1):221-64. PMID:    21248167.-   [3]. Zgheib C1, Xu J1, Liechty KW1. Targeting Inflammatory Cytokines    and Extracellular Matrix Composition to Promote Wound Regeneration.    Adv Wound Care (New Rochelle). 2014 Apr. 1; 3(4):344-355. PMID:    24757589.-   [4]. Voelcker V1, Gebhardt C, Averbeck M, Saalbach A, Wolf V, Weih    F, Sleeman J, Anderegg U, Simon J. Hyaluronan fragments induce    cytokine and metalloprotease upregulation in human melanoma cells in    part by signalling via TLR4. Exp Dermatol. 2008 February;    17(2):100˜7. PMID: 18031543.-   [5]. Esser PR1, Wölfle U, Dürr C, von Loewenich F D, Schempp C M,    Freudenberg M A, Jakob T, Martin S F. Contact sensitizers induce    skin inflammation via ROS production and hyaluronic acid    degradation. PLoS One. 2012; 7(7):e41340. PMID: 22848468.-   [6]. Black KE1, Collins S L, Hagan R S, Hamblin M J, Chan-Li Y,    Hallowell R W, Powell J D, Horton M R. Hyaluronan fragments induce    IFNβ via a novel TLR4-TRIF-TBK1-IRF3-dependent pathway. J Inflamm    (Lond). 2013 May 30; 10(1):23. PMID: 23721397.-   [7]. Horton MR1, McKee C M, Bao C, Liao F, Farber J M, Hodge-DuFour    J, Puré E, Oliver B L, Wright T M, Noble P W. Hyaluronan fragments    synergize with interferon-gamma to induce the C-X-C chemokines mig    and interferon-inducible protein-10 in mouse macrophages. J Biol    Chem. 1998 Dec. 25; 273(52):35088-94. PMID: 9857043.-   [8]. Hodge-Dufour J1, Noble P W, Horton M R, Bao C, Wysoka M,    Burdick M D, Strieter R M, Trinchieri G, Puré E. Induction of IL-12    and chemokines by hyaluronan requires adhesion-dependent priming of    resident but not elicited macrophages. J Immunol. 1997 Sep. 1;    159(5):2492-500. PMID: 9278343.-   [9]. McKee CM1, Lowenstein C J, Horton M R, Wu J, Bao C, Chin B Y,    Choi A M, Noble P W. Hyaluronan fragments induce nitric-oxide    synthase in murine macrophages through a nuclear factor    kappaB-dependent mechanism. J Biol Chem. 1997 Mar. 21;    272(12):8013-8. PMID: 9065473.-   [10]. McKee CM1, Penno M B, Cowman M, Burdick M D, Strieter R M, Bao    C, Noble P W. Hyaluronan (HA) fragments induce chemokine gene    expression in alveolar macrophages. The role of HA size and CD44. J    Clin Invest. 1996 Nov. 15; 98(10):2403-13. PMID: 8941660.-   [11]. Ghosh S1, Hoselton SA2, Wanjara SB2, Carlson J3, McCarthy JB4,    Dorsam GP2, Schuh JM2. Hyaluronan stimulates ex vivo B lymphocyte    chemotaxis and cytokine production in a murine model of fungal    allergic asthma. Immunobiology. 2015 Feb. 7. PMID: 25698348.-   [12]. Ghosh S1, Samarasinghe A E, Hoselton S A, Dorsam G P, Schuh    J M. Hyaluronan deposition and co-localization with inflammatory    cells and collagen in a murine model of fungal allergic asthma.    Inflamm Res. 2014 June; 63(6):47˜84. PMID: 24519432.-   [13]. Nikitovic D1, Berdiaki A2, Galbiati V3, Kavasi RM2, Papale A3,    Tsatsakis A4, Tzanakakis GN2, Corsini E3. Hyaluronan regulates    chemical allergen-induced IL-18 production in human keratinocytes.    Toxicol Lett. 2014 Oct. 1; 232(1):89-97. PMID: 25280773.-   [14]. Fieber C1, Baumann P, Vallon R, Termeer C, Simon J C, Hofmann    M, Angel P, Herrlich P, Sleeman J P.    Hyaluronan-oligosaccharide-induced transcription of    metalloproteases. J Cell Sci. 2004 Jan. 15; 117(Pt 2):359-67. PMID:    14657275.-   [15]. Campo GM1, Avenoso A, D'Ascola A, Scuruchi M, Prestipino V,    Nastasi G, Calatroni A, Campo S. The inhibition of hyaluronan    degradation reduced pro-inflammatory cytokines in mouse synovial    fibroblasts subjected to collagen-induced arthritis. J Cell Biochem.    2012 June; 113(6):1852-67. PMID: 22234777.-   [16]. Campo GM1, Avenoso A, D'Ascola A, Prestipino V, Scuruchi M,    Nastasi G, Calatroni A, Campo S. 4-mer hyaluronan oligosaccharides    stimulate inflammation response in synovial fibroblasts in part via    TAK-1 and in part via p38-MAPK. Curr Med Chem. 2013; 20(9):1162-72.    PMID: 23298137.-   [17]. Liang J1, Jiang D, Jung Y, Xie T, Ingram J, Church T, Degan S,    Leonard M, Kraft M, Noble P W. Role of hyaluronan and    hyaluronan-binding proteins in human asthma. J Allergy Clin Immunol.    2011 August; 128(2):403-411. PMID: 21570715.

However, the applicant's research achievements in recent years (patentapplication No. 200780052196.7, 201310325056.X, 201310454955.X,201410153593.5, 201510065499.9, 201510065498.4, 201510067326.0 and201510333526.6) suggest another possibility that LMW-HA fragmentsin-vitro preparation have the function of inhibiting inflammation ofskin and mucous membranes. According to these references, the role ofthe LMW-HA fragments in inhibiting inflammation may be related either tothe extent of molecular size or to its manufacture method. Moreover, thecomparative results of experiments on small animals, large animals andhuman also show that the function of the LMW-HA fragments may becompletely different in small animals, large animals and human. Forexample, the above patent references indicated that the LMW-HA fragmentswith a molecular weight distribution of 10˜60 KD could be prepared intoin vitro preparations for topical use (such as daily necessities,hygiene products, cosmetics, skin care products, disinfecting products,etc.) to help alleviate or treat inflammation of skin and mucousmembranes; whereas other's LMW-HA less than 10 KD substantially did nothave such effect. However, according to the recent patent WO/2014/165713of CEDARS-SINAI MEDICAL CENTER, LMW-HA less than 10 KD produced by useof streptococci hyaluronidase digesting HA did not induce aninflammatory reaction, but LMW-HA greater than 10 KD produced byStreptomyces hyaluronidase induced an inflammatory reaction, suggestingthat the function of LMW-HA may also be related to the type of enzymefor digestion, namely, manufacture methods which lead to differences inthe three-dimensional structure or molecular arrangement of HAfragments.

These contradictory research results suggest that it is difficult todetermine the exact function of LMW-HA fragments at present. Perhaps theproduction mode, molecular weight, type of enzyme for digestion, themode of use, etc. of LMW-HA fragments may affect their functionalperformance, but in addition to these known possible factors, there aremany other unknown factors for the researchers currently, for example,reliable experimental results of large animals are not available.Therefore, although the insiders pin their hope on the medical value ofLMW-HA, it is difficult to predict the clinical research and success ofLMW-HA, and it is quite difficult to determine the clinical indications.A feasible and effective LMW-HA in vitro preparation has been obtainedin previous researches, but due to the complex internal environment ofhuman, the LMW-HA in vivo injection may be completely different from thein vitro preparation, whereas the role may not be the same in human,large animals and small animals. Therefore, it requires for furtherverification by clinical studies. In addition, LMW-HA used for in vivoinjection will have higher requirements on more aspects, for example, itshall meet the requirements of high purity, no severe pain wheninjection or after injection and no allergic reaction, and theindications and side effects, as well as the dose of each injection, theway of injection, etc. shall be specified. It will have great scientificsignificance and application value in solving the above unpredictableproblems.

SUMMARY

The invention aims to provide the application of LMW-HA fragments in thefollowing aspects, including: The application of the LMW-HA fragments inpreparing drugs for treating solid tumors.

Specifically, the solid tumors include, but are not limited to, lungcancer, pancreatic cancer, oral cancer, meningioma, gastric cancer andovarian cancer.

The application of the LMW-HA fragments in preparing drugs for treatingxerophthalmia, muscae volitantes, conjunctival diseases, vitreousopacity, and vitreous detachment or impaired vision caused by vitreousopacity.

The application of the LMW-HA fragments in preparing drugs for treatingmyofascitis, muscle and tendon injury, hyperosteogeny, bone spur,protrusion of intervertebral disc and diseases of lumbar spinal diseasesand cervical vertebra.

The application of the LMW-HA fragments in preparing drugs for treatinggout, arthritis and ankylosing spondylitis.

The application of the LMW-HA fragments in preparing drugs for treatingcardiovascular diseases.

The application of the LMW-HA fragments in preparing drugs for promotingthe functional rehabilitation after cerebral infarction or cerebralhemorrhage.

The application of the LMW-HA fragments in preparing drugs for treatingdysmenorrheal, endometriosis and adenomyosis.

The application of the LMW-HA fragments in preparing drugs for treatingperiodontal diseases, dental ulcer and guttural diseases.

The application of the LMW-HA fragments in preparing drugs for treatingherpes zoster, neurodermatitis, psoriasis, eczema, eczema herpeticum andpompholyx.

The application of the LMW-HA fragments in preparing drugs for treatingburn, trauma and surgical wound.

The application of the LMW-HA fragments in preparing drugs for treatingpain or pruritus.

The application of the LMW-HA fragments in preparing drugs for treatingacute pancreatitis.

The application of the LMW-HA fragments in preparing drugs for treatingabdominal mucous membrane adhesion and pelvic mucous membrane adhesionafter operation or peritoneal dialysis.

The application of the LMW-HA fragments in enhancing or restoringphysical strength and energy after chemotherapy or in preparing drugsfor treating side effects after chemotherapy.

The application of the LMW-HA fragments in facial cosmetology or inpreparing drugs for treating facial aging.

The application of the LMW-HA fragments in preparing drugs for reducingsubcutaneous fat tissue.

The above LMW-HA fragments are HA fragments with a molecular weightdistribution of 10˜60 KD, and the HA fragments are obtained from HMW-HAas raw material through recombinant human hyaluronidase PH20 afterdigestion.

Further, the recombinant human hyaluronidase PH20 is the recombinanthuman hyaluronidase produced by zooblasts or yeast or plant expression,with the purity higher than 98.5%.

Further, the LMW-HA fragments are prepared into an injection forapplication, and the injection contains an effective dose of HAfragments with a molecular weight distribution of 10˜60 KD.

Further, the injection is injected via abdominal subcutaneous fat layeror at painful/itchy points or diseased sites.

Further, the injection is administrated 1-2 times per day, and theeffective dose of HA fragments is 100˜200 mg per injection.

Further, the injection is a liquid preparation, which is prepared by thefollowing steps: A. Prepare HMW-HA raw material into solution, introducesodium chloride and magnesium ions, and add recombinant humanhyaluronidase PH20 for digestion to obtain the injection containing HAfragments with a molecular weight distribution of 10˜60 KD; B.Inactivate the residual recombinant human hyaluronidase PH20 in theobtained injection; C. Inactivate viruses and remove bacteria through0.22 μm filtration or inactivate bacteria.

Further, the HMW-HA raw material in Step A is baked at 105° C. for 5˜6 hand 120° C. for 1˜2 h respectively in advance, and they are thenprepared into solution.

Further, the HMW-HA raw material in Step A has a molecular weightdistribution of 800˜1,200 KD. 4,000˜5,000 units of the recombinant humanhyaluronidase PH20 are added to each 1 g of the HMW-HA raw materialcorrespondingly for digestion, and the enzyme digestion reaction is keptfor 5˜6 h.

Further, the HMW-HA raw material in Step A is directly prepared intosolution, the HMW-HA raw material has a molecular weight distribution of800˜1,200 KD. 15,000˜20,000 units of the recombinant human hyaluronidasePH20 are added to each 1 g of the HMW-HA raw material correspondinglyfor digestion, and the enzyme digestion reaction is kept for 5˜6 h.

Further, the concentrations of sodium chloride and magnesium ionsintroduced in Step A in the prepared solution are 80˜90 mM and 1 mM,respectively.

Further, the inactivated recombinant human hyaluronidase is also removedafter Step B.

Further, each 2˜4 ml of the injection contains 100 mg of HA fragments,and the residual recombinant human hyaluronidase shall not exceed 20 ug.

Further, the final concentration of magnesium ions in the injection is 1mM, and the final concentration of sodium chloride in the injection is115˜125 mM.

Owing to the above technical schemes, the invention at least has thefollowing advantages:

-   (1) Based on the experimental studies on the LMW-HA fragments for    small animals, large animals and human, the invention finds that    such HA fragments have a series of new applications in injection    use. The experimental results show that the LMW-HA fragments have    unexpected therapeutic effects on multiple diseases, as well as the    efficacies of relieving pain and itching rapidly and enhancing    physical strength and energy. Thus the LMW-HA fragments have great    application values in preparing drugs or adjuvant drugs for treating    relevant diseases. Besides, according to the experimental results,    it can reduce subcutaneous fat, especially that of human head, face    and neck; at the same time, it also has the efficacies on facial    cosmetology and anti-aging, it can be injected via abdominal    subcutaneous fat layer instead of facial injection, which will not    result in pain and facial inflammations. Therefore, the LMW-HA    fragments also have outstanding application potential in the fields    of body care, anti-aging and cosmetology.-   (2) The LMW-HA fragments for injection use can be prepared into a    form of liquid preparation. Due to the convenient production and    stable preparation, it is applicable for batch production, and no    allergic reaction and side effect have occurred for human injection    while in use.

BRIEF DESCRIPTION OF THE DRAWINGS

The above are only the summarization on the technical schemes of theinvention. In order to get clearer understanding about the technicalmeans, the invention will be further explained in details by combiningwith drawings and embodiments.

FIG. 1 is an electropherogram of high-purity glycosylated recombinanthuman hyaluronidase derived from CHO cells of the shaking culturebioreactor.

FIG. 2 is a bottled injection of HA fragments.

FIG. 3 shows the antibacterial effect of HA fragments with a molecularweight distribution of 10 KD-60 KD (1% B-HA in the figure means theinjection treatment group treated by the injection containing 1% HAfragments with a molecular weight distribution of 10 KD-60 KD).

FIG. 4 shows the anti-inflammatory and antibacterial effects of theinjection of the invention on the skin wound of small animal (B-HA inthe figure means the injection treatment group treated by HA fragmentswith a molecular weight distribution of 10 KD-60 KD).

In FIG. 5A-FIG. 5E show the effects of LMW-HA fragments injection onIL-1, IL-6, IFNβ, IFN-α and IL-10, respectively (in the figure:LPS=endotoxin; L+A=LPS lipopolysaccharide+anti-human TLR4 single chainantibody; L+HA=LPS lipopolysaccharide+HA fragments with a molecularweight distribution of 10 KD-60 KD).

FIG. 6 shows the ophthalmological laboratory diagnostic result of asubject suffering from vitreous opacity and vitreous detachment.

FIG. 7 shows the comparison of pictures of partial subjects before andafter the treatment with injection of LMW-HA fragments via abdominalsubcutaneous fat layer, indicating that the injection of LMW-HAfragments has a therapeutic effect on human facial cosmetology andfacial anti-aging.

FIG. 8 shows the comparison before and after the treatment withinjection of LMW-HA fragments for a case of extensive burns.

FIG. 9 shows the effect after the treatment with injection of LMW-HAfragments via abdominal subcutaneous fat layer for a case ofneurodermatitis (the focus is indicated by arrow in the picture).

FIG. 10 shows the effect before and after the treatment with injectionof LMW-HA fragments via abdominal subcutaneous fat layer for a case ofpsoriasis.

FIG. 11 shows the enhanced MRI reports before and after the treatmentwith injection of LMW-HA fragments for a case of meningioma.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As mentioned above, based on the current industrial researchachievements, it is difficult to determine the exact functions ofLMW-HA. And there are even contradictive functional phenomena accordingto the results of current scientific experiments, whereas the mechanismof action for such phenomena is still completely unclear. On the basisof previous researches (patent application No. 200780052196.7,201310325056.X, 201310454955.X, 201410153593.5, 201510065499.9,201510065498.4, 201510067326.0 and 201510333526.6), the invention hopesto further explore the unknown functions of LMW-HA fragments bydesigning studies on small animals, large animals and human. Accordingto the experimental studies on small animals and large animals, theinventor finds that the LMW-HA fragments in injection use have uncleartherapeutic effect on species of small animals but it has a clearanti-inflammatory effect on large animals. At the same time. the resultsof clinical studies on human also unexpectively suggest that the LMW-HAfragments have a series of new applications in injection use, and suchapplications can be reflected by more convenient ways of injection (suchas injection via abdominal subcutaneous fat layer or at painful/itchingpoint or diseased sites).

The experimental results of clinical studies show that the LMW-HAfragments have an unexpected effect of treating multiple diseases oralleviating symptoms in injection use. Specifically, the multiplediseases include, but are not limited to, solid tumors (such as lungcancer, pancreatic cancer, oral cancer, meningioma, gastric cancer,ovarian cancer, etc.), xerophthalmia, muscae volitantes, conjunctivaldiseases, vitreous opacity, vitreous detachment or impaired visioninduced by vitreous opacity, myofascitis, muscle and tendon injury,hyperosteogeny, bone spur, protrusion of intervertebral disc, diseasesof lumbar spinal diseases and cervical vertebra, gout, arthritis,ankylosing spondylitis, cardiovascular diseases (including but notlimited to coronary heart disease and the complications afterintravascular stent), dysmenorrhea, endometriosis, adenomyosis,periodontal disease, dental ulcer and guttural diseases, herpes zoster,neurodermatitis, psoriasis, eczema, eczema herpeticum, pompholyx, burn,trauma, surgical wound, etc., as well as abdominal mucous membraneadhesion, pelvic mucous membrane adhesion, etc. induced after operationor peritoneal dialysis; besides, it can prevent the transfer of solidtumors, accelerate the functional rehabilitation after cerebralinfarction or cerebral hemorrhage, enhance or recover physical strengthand energy after chemotherapy or treat the side effects afterchemotherapy, and it can also quickly alleviate pain or pruritus;according to these results, LMW-HA fragments have a great applicationvalue in preparing drugs or adjuvant drugs for treating relevantdiseases.

Besides, the experimental results also suggest that LMW-HA fragmentsreduce subcutaneous fat, especially that of human head, face and neck.At the same time, they also have the efficacies on facial cosmetologyand anti-aging. The existing HMW-HA in the market is used by facialinjection, and it is injected into dermal wrinkles and concaves or thesite to be smoothed in the form of filler to realize the application forfacial cosmetology, such as filling concaves, removing wrinkles,moulding, etc.; the LMW-HA fragments in the invention differ in thatthey can be injected via abdominal subcutaneous fat layer while in useinstead of facial injection, which will not result in pain and facialinflammation. Therefore, the LMW-HA fragments also have an outstandingapplication potential in the fields of body care, anti-aging andcosmetology.

The LMW-HA fragments of the invention in the above experiments areobtained by the following specific manufacture method: the LMW-HAfragments are HA fragments with a molecular weight distribution of 10˜60KD obtained from HMW-HA digested directly by recombinant humanhyaluronidase PH20. In the process of production, recombinant humanhyaluronidase PH20 can be produced by Chinese hamster ovary (CHO) cellsor yeast or plant expression, for example, it has been recorded in theprevious patent application documents (publication No.: CN104342420A,CN103468662A and CN105018547A) that glycosylated recombinant humanhyaluronidase PH20 is produced by using the Chinese hamster ovary (CHO)cells.

In order to ensure the safety in injection use, high purity is preferredfor recombinant human hyaluronidase PH20 (preferred with apurity>98.5%), and a medical purity>98.5% is preferred for HMW-HA rawmaterial. In practical production, raw material of high purity can beused directly, or recombinant human hyaluronidase PH20 and HMW-HA rawmaterial of low purity can also be used for the subsequent steps afterpurification. Besides, on completion of the enzyme digestion reaction,recombinant human hyaluronidase shall also be inactivated, following byinactivating viruses and filtering or inactivating bacteria. Bycombining with the results of clinical studies, it has been found thatHA fragments containing protein residue of recombinant humanhyaluronidase have no allergic reaction and side effect in injectionuse, but it is suggested to separate and remove the protein residue ofrecombinant human hyaluronidase PH20 after inactivation.

For convenience of injection use, LMW-HA fragments are prepared into aliquid injection for use during the experiments, and the injectioncontains an effective dose of HA fragments with a molecular weightdistribution of 10˜60 KD. In practical application, the LMW-HA fragmentsfor injection use can be prepared into other forms of injection, forexample, they can also be prepared into sterile powder or concentratedsolution which will be mixed into solution before use.

The invention will be explained through the specific embodiments below.It shall be understood that the embodiments described here are only fordescribing and explaining the invention but not for limiting theinvention. Besides, unless otherwise specified, the drugs or reagentsinvolved in the following embodiments can all be purchased from normalbusiness channel. Unless otherwise specified, the experimentaltechniques or operating methods involved in the following embodimentsall refer to the known conventional experimental techniques or operatingmethods in this field.

Embodiment 1

Objective: To produce recombinant human hyaluronidase PH20 and prepareLMW-HA fragments injection

Methods: Based on the methods recorded in the patent documents No.CN104342420A, CN103468662A and CN105018547A, glycosylated recombinanthuman hyaluronidase PH20 was produced using Chinese hamster ovary (CHOcells), including: Artificially synthesize the gene cDNA of glycosylatedrecombinant human hyaluronidase PH20, insert it into the empty GC-richexpression vectors of pMH3, pMH4 and pMH5 to construct the expressionvectors of pMH3-PH20, pMH4-PH20 and pMH5-PH20 (the expression vectorswere constructed using the methods recorded in the patent document No.CN102124019A so as to extremely highly express recombinant protein);then transfer the cDNA expression vectors of pMH3-PH20, pMH4-PH20 andpMH5-PH20 into the CHO-S cell line, and screen the CHO-S cell clones forcell lines that express the highest level of human hyaluronidase PH20.Then, the cell lines that express the highest level of humanhyaluronidase PH20 was scaled up for large-scale culture in the shakingcell culture bioreactor; filter the harvested condition mediumcontaining PH20 through 0.22 μm filtration membrane, then separate by2-3 steps using ion column, hydrophobic column and hydroxyapatite columnetc., then perform the steps of bacterial filtration, virus filtrationand inactivation, ultrafiltration for preparation of concentratedpurified bulk of sterile and virus-free glycosylated recombinant humanhyaluronidase PH20 with a purity>99.0%.

Use an enzyme digestion and blending bioreactor with a working volume of25 L featured with cleaning and sterilization in-place, and prepareinjection-grade HA raw material with a molecular weight distribution of800˜1,200 KD; add and blend the injection-grade HA raw material with amolecular weight distribution of 800˜1,200 KD into injection-grade purewater for intensive dissolution one time or several times; then addsodium chloride, magnesium ions and recombinant human hyaluronidaseuntil reaching their final concentration of 80˜90 mM, 1 mM and15,000˜20,000 units (about 15˜20 μg) per 1 g of HMW-HA respectively inorder, mix them completely, and react at 37° C. for 5˜6 h until that themolecular weight of HA fragments reaches 10 KD˜60 KD. Add sodiumchloride at a final concentration of 35˜45 mM to regulate the osmoticpressure to 280˜300 mOsm/L, then heat it at 84˜95° C. for 30˜60 min(thermal inactivation of recombinant human hyaluronidase, partialbacterial inactivation and virus inactivation (by simultaneouslyreducing the pH level), and then remove bacteria by 0.22 μm filtration.Solution 1 was obtained after the filtration, and it contained theresidue of inactivated recombinant human hyaluronidase. Further, removethe protein residue of inactivated recombinant human hyaluronidase PH20in Solution 1 by using affinity column chromatography, precipitation,ultrafiltration and dialysis in order or respectively to obtain Solution2 containing 10 KD-60 KD HA fragments, sodium chloride and magnesiumions only.

In order to research the production method of injection preparation andclearly know the effect of recombinant human hyaluronidase afterinactivation, two injections containing HA fragments were obtainedaccording to the above production method in the embodiment: one based onSolution 1 contains the protein residue of inactivated recombinant humanhyaluronidase PH20 without enzyme activity (the amount of the proteinresidue shall be controlled in production practice for safety andreliability in use). The other one based on Solution 2 does not containthe protein residue of inactivated recombinant human hyaluronidase PH20.

Results.

The glycosylated recombinant human hyaluronidase with a purity>99% andproduced from CHO cells was obtained (FIG. 1 );

The following two injections containing LMW-HA fragments were prepared,and the injections were filled in small bottles and prepared into HAfragments with an effective dose of 100 mg per bottle (FIG. 2 ). Afterfinal filling and packaging, the injections can be stably stored at4˜12° C. or −8˜−70° C. or room temperature for 2 years.

Injection 1: Each 2˜4 ml contained 100 mg of HA fragments with amolecular weight distribution of 10 KD˜60 KD and <20 μg of proteinresidue of inactivated recombinant human hyaluronidase PH20 withoutenzyme activity (endotoxin<0.5 IU/ml, sterile, virus-free), the finalconcentration of sodium chloride was 115˜125 mM, and the finalconcentration of magnesium ions was 1 mM.

Injection 2: Each 2˜4 ml contained 100 mg of HA fragments with amolecular weight distribution of 10 KD˜60 KD but did not contain proteinresidue of inactivated recombinant human hyaluronidase PH20(endotoxin<0.5 IU/ml, sterile, virus-free), the final concentration ofsodium chloride was 115˜125 mM, and the final concentration of magnesiumions was 1 mM.

Conclusion: The results suggest that HA fragment Injection 1 containingprotein residue of recombinant human hyaluronidase and Injection 2 notcontaining protein residue of recombinant human hyaluronidase aresuccessfully prepared, and the production technology for the injectionof HA fragments is established.

Besides, it has been found in the following experiments that the twoforms of injections have basically the same therapeutic effect, and haveno allergic reaction and other side effects (which meets the FDAstandards for biological medicine and Type III medical devices),suggesting that inactivated recombinant human hyaluronidase PH20 has noinfluence on the effect and safety of injection of HA fragments. HAfragments with a molecular weight distribution of 10 KD-60 KD is themain active ingredient in the injections, and such bioactive HAfragments (the LMW-HA fragments) are called as B-HA for short.

Embodiment 2

Objective: To study bacterial inhibition activity, in-vitro cellularactivity, and activity in small animals for the above HA fragmentinjections (Injection 1 and Injection 2).

Methods: Dilute Injection 1 and Injection 2 with normal saline to doublevolume for study bacterial inhibition activity, in-vitro cellularactivity, and activity in small animals of the above HA fragmentinjections (Injection 1 and Injection 2).

Results: FIG. 3 showed that Injection 1 inhibited the cloning and growthof Porphyromonas gingivalis, suggesting that the HA fragment injectionwas not favorable for the growth of Porphyromonas gingivalis (note: thenegative control was normal saline, and the positive control wasantibiotic metronidazole);

FIG. 4 showed that Injection 1 inhibited the cloning and growth ofPorphyromonas gingivalis in the in vivo experiment on mice, suggestingthat the HA fragment injection was not favorable for the growth ofPorphyromonas gingivalis at the epidermal wound of small animals;

FIGS. 5A-5E showed that Injection 1 significantly enhanced IL-10secretion and inhibited the secretions of TNF, IL-1, IL-6 and interferonbeta, which were induced by endotoxin (LPS), of human TLR4 receptorpositive cells. The results of Injection 2 (data not shown) were similarto the above results.

Conclusion:

1. The above HA fragment injections (Injection 1 and Injection 2) havesignificant anti-inflammatory effect on in-vitro cultured human cells,and one of the main mechanisms of action may be that it accelerates thesecretion of the anti-inflammatory cytokine IL-10, suggesting that B-HAis different from common HA. Although it has the same chemicalcomposition of HA, it may combine with TLR4 by its different3-dimensional molecular arrangements so as to antagonize endotoxin,accelerate the secretion of the anti-inflammatory cytokine IL-10, and torealize the actions of anti-inflammation, including relieving pain anditching, and effectively treating various diseases and morbid states; ofcourse, some studies (including: Cuixia Yang, Manlin Cao, Hua Liu,Yiqing He, Jing Xu, Yan Du, Yiwen Liu, Wenjuan Wang, Lian Cui, JiajieHu, Feng Gao, The high and low molecular weight forms of hyaluronan havedistinct effects on CD44 Clustering, Published on Nov. 1, 2012 asManuscript M112.349209) also suggested that the therapeutic mechanism ofHA fragments may also be involved in the regulation of inflammatory cellmigration into inflamed tissues which is mediated by CD44 receptor. Thisis a new action mechanism of LMW-HA fragments for treating variousdiseases and morbid states, as well as serves as an activity assay fordetection of B-HA anti-inflammation bioactivity.

2. The above HA fragment injections (injection 1 and Injection 2) havebacterial growth inhibiting or antimicrobial activities both in vivo andin vitro.

Embodiment 3

Background: The research in Embodiment 2 indicates that B-HA may realizethe actions of anti-inflammation, relieving pain and itching, andeffectively treating various diseases and morbid states by a new actionmechanism of accelerating the secretion of anti-inflammatory cytokineIL-10 through interaction with TLR4. The action mechanism provides atheoretical foundation for B-HA in the unexpectedly effective treatmentof various diseases and morbid states.

Objective: To study the effects of the above HA fragment injections(Injection 1 and Injection 2) on inflammation in small animals and largeanimals.

Research Background: Research literatures show that the bioactivity orfunction of HA or its fragments is not only related to the molecularweight and/or the manufacture method, but also to the species ofanimals. The studies in small animals such as mice suggest that thebioactivity of HA fragments (LMW-HA) promotes inflammations, but thestudies in large animals and human suggest that the bioactivity of HAinhibits inflammations. Therefore, different species of animalsincluding small and large are employed in this embodiment to study thebioactivity of the above HA fragment injections (Injection 1 andInjection 2) on different types of inflammation. The resultsdemonstrated that HA fragment injection has an anti-inflammatoryactivity in large animals.

3.1 Bioactivity of HA Fragment Injection (B-HA) in Inflammatory CervicalWound, Spinal Cord Wound and Axonal Sprouting of Small Animal Mice.

A total of 10 female ICR mice were used in this experiment. Dissect thecervical spinal cord and cut it off with a 11 # scalpel. Protect the cutspinal cord wounds with PEG MW 400 cell membrane fusogen, then suturethe muscle and skin, and provide nutrition via caudal veins of the mice4 times a day using venous high-nutrient normal saline and HA fragmentinjection+venous high-nutrient normal saline. After 4 weekspostoperatively, all the mice had partial neurobehavioral recovery, andthen all the mice were killed. Cryopreserve the cut spinal cord woundsusing dry ice and perform cryosection, then fix them with 4% formalinand perform immunohistochemical staining using Anti-neurofilament 200and DAPI, and examine axonal sprouting using a fluorescent or confocalmicroscope.

Results: Table 1 showed the inflammations of cervical wounds, the axonalsprouting on the surface and the wound healing of spinal cord wound inthe mice treated by the above HA fragment Injection 1 and control mice.The situations of Injection 2 were basically the same as those ofInjection 1 without significant differences, and those was not shownhere.

TABLE 1 Inflammations of Cervical Wound, Axonal Budding on the Surfaceof Spinal Cord Wound and Wound Healing in the Mice Axonal Sprouting onthe Control Number Surface of Conditions of of Spinal Cord InflammationsWound Samples Wound of Wound Healing Venous High 5 + + + Nutrition +Normal Saline 1% B-HA + 5 + + + Venous High Nutrition + Normal SalineNote: Axonal sprouting on the surface of spinal cord wound was judged as−, + and ++, representing no sprouting, sprouting and obvious sprouting,respectively; the control conditions of inflammations of wound werejudged as −, + and ++, representing that the inflammation was poorlycontrolled, properly controlled and perfectly controlled, respectively;wound healing was judgd as −, + and ++, representing that the woundhealing was quite abnormal, normal, and obviously better than normal,respectively.

Conclusion: The above HA fragment injections (Injection 1 and Injection2) cannot significantly control the inflammation of cervical wound andcannot significantly promote the axonal sprouting and wound healing ofspinal cord wound in mice, indicating that the HA fragment injectionshave no obvious anti-inflammatory effect on mice as a species of smallanimals.

3.2. A Study of Controlling Myocardial Inflammation in Mice.

Ten C57/BL6 mice of age 6-week were used in this experiment, and micemodels of myocardial inflammation were produced using angiotensin II(1,500 ng/kg per minute) for 7 consecutive days. With 5 mice in eachgroup, Group 1 was injected with HA fragment injection+normal saline,and Group 2 was injected with normal saline. After 7 days, heartsections were stained with hematoxylin, eosin and Masson trichrome forhistochemical staining to observe the infiltration of myocardialinflammatory cells.

Results: Table 2 showed the effect of HA fragment Injection 1 and normalsaline on the myocardial infiltration of inflammatory cells of the mice.The effect of Injection 2 were basically the same as those of Injection1 without significant differences, and those were not shown here.

TABLE 2 Myocardial Infiltration of Inflammatory Cells in Mice NumberInfiltration of of Inflammatory Samples Cells Normal Saline 5 + 1%B-HA + Normal Saline 5 + + Note: The myocardial infiltration ofinflammatory cells was judged as −, + and ++, representing that theinfiltration of inflammatory cells was not obvious, obvious and quiteobvious, respectively.

Conclusion: The above HA fragment injections (Injection 1 and Injection2) significantly promote the infiltration of myocardial inflammatorycells induced by angiotensin II in mice, indicating that the HA fragmentinjections have the bioactivity in promoting inflammation in mice as aspecies of small animals.

3.3 Bioactivity of B-HA (HA Fragment Injection 1) on Abdominal Adhesionin Dogs.

Six healthy beagle dogs were used in this experiment, weighing 6.8±0.8kg. After 7 days of pre-feeding, the 6 dogs were randomly divided intoblank control group (n=2), B-HA low-dose group (n=2) and B-HA high-dosegroup (n=2). The experimental dogs were fasted for 12 h and deprived ofwater for 6 h before operation. At 15 min before operation, the dogs inthe high-dose group were subcutaneously injected with the above HAfragment injection 0.8 ml/kg on the back; the dogs in the low-dose groupwere subcutaneously injected with the above HA fragment injection 0.4m/kg on the back; the dogs in the blank group were not injected. Performanesthetization and conventional preparation of operative site, and fixthem in a supine position, conventionally open the abdominal cavityalong the midline of abdomen at 2 cm behind the umbilicus, search thececum and find the ileocecal junction, take the ileum at about 20 cmfrom the ileocecal junction out of the abdominal cavity, isolate andconventionally cut off the intestinal canal, and perform end-to-endanastomosis in a full-thickness continuous suture. On completion ofsuturing, inject normal saline into the anastomotic part, check theairtightness of the anastomotic part, apply 2 ml of the above HAfragment injection at the operative site in the low-dose group and thehigh-dose group, and then suture the abdominal wall layer by layer.Record the operative practice in details during the operation. The dogswere fasted for 3 days and were then given a liquid diet (addingappropriate oral liquid in yogurt for salt supplementation) withincreasing amounts, the animals were fed with soft dog food afterdefecation and gradually transited to normal dog food, and postoperativeobservation was performed every morning for 7 consecutive days.

Results: 1. Postoperative observation: The postoperative conditions ofthe dogs were observed, and it was found that in the untreated blankgroup, the mental state and activity state were significantly worse thanthose in the experimental groups within 2 days postoperatively, feveroccurred within 4 days postoperatively, and the body temperature, mentalstate and activity state recovered to the preoperative levels from Day 5postoperatively to the end of monitoring; 2. In the HA fragmentinjection low-dose and high-dose groups, the postoperative mental stateand activity state were not significantly different from thosepostoperatively, indicating that the HA fragment injection effectivelycontrolled the postoperative fever, abdominal inflammation and paininduced by system inflammatory factors, it did not affect the activityand promoted the recovery of intestinal peristalsis, and it reduced thepossibility of abdominal adhesion; 3. At Day 7 postoperatively,exploratory laparotomy was performed in each group, there was no woundhealing disorder by visual inspection, such as intestinal anastomoticfistula and intestinal stenosis or obstruction. In the blank group, theadhesion of operative site with greater omentum was observed. In the HAfragment injection low-dose and high-dose groups in Embodiment 1, theadhesion of operative site with greater omentum was not observed. Localadhesion of surgically wounded site with adjacent intestinal tract couldbe observed in all the blank group and the HA fragment injectionlow-dose and high-dose groups, and it was speculated that the localadhesion of surgically wounded site with adjacent intestinal tract couldbe effectively obstructed by the gel physical method of high-viscosityHA fragment.

TABLE 3 Experimental Results of HA Fragment Injection 1 in Preventingand Treating Abdominal Adhesions of Dogs. Adhesion of intestinal Post-anastomotic operative Number Postoperative body stoma with wound oftemperature, mental greater healing samples state and activity stateomentum disorder Blank Group 2 Obviously poor mental Yes No (untreated)state, inactivity, high fever as compared with those preoperativelyLow-dose HA 2 No significant No No Fragment difference in body Injectiontemperature, mental state and activity state as compared with thosepreoperatively High-dose HA 2 No significant No No Fragment differencein body Injection temperature, mental state and activity state ascompared with those preoperatively

Conclusion. The subcutaneous injection of HA fragment injection(Injection 1) and administration by applying on the operative site cansignificantly improve fever, abdominal inflammation and pain induced byreleased systemic inflammation factors after intestinal anastomosisoperation in dogs. Besides, these therapeutic actions do not affectphysical activities and promote the recovery of intestinal peristalsis,and reduce possible abdominal adhesion. In addition, side effectsincluding wound healing disorders are not observed, such as intestinalanastomotic fistula and intestinal stenosis or obstruction. Resultsindicate that the above HA fragment injections play a role in preventingand treating local abdominal adhesions by their anti-inflammatory actionin dogs as a species of large animals.

3.4. Effect of HA Fragment Injections in Controlling Skin WoundInflammation in Dogs.

Six healthy beagle dogs were used, weighing 6.8±0.8 kg. After 7 days ofpre-feeding, the 6 dogs were randomly divided into blank control group(n=3) and HA fragment injection treatment groups (n=3). The experimentaldogs were fasted for 12 h and deprived of water for 6 h preoperatively.At 15 min preoperatively, the dogs in the HA fragment Injection 1treatment group 1 and the HA fragment Injection 2 treatment group 2 wereinjected subcutaneously with 0.8%/kg normal saline containing 1% B-HA,and those in the blank group were injected subcutaneously with normalsaline. Perform anesthesia and conventional preparation of operativesite, incise for 3 cm on both sides of the midline on the back withoutsuture, and contaminate the wound with contaminated soil. Afteroperation, the dogs in the treatment groups 1 and 2 were injectedsubcutaneously with 0.8 ml/kg HA fragment injection+normal saline on theback every day, whereas those in the blank group were injectedsubcutaneously with normal saline on the back every day, andpostoperative observation was performed every morning for 7 consecutivedays.

Results: See Table 4.

TABLE 4 Effect of HA Fragment Injections in the Back Wound Inflammationof Dogs. Number Period of Situation of Size of back of inflammation backwound wound after samples (days) inflammation incrustation HA Fragment 3 5 Quite unobvious Small Injection 1 Experimental Group 1 HA Fragment 3 5 Quite unobvious Small Injection 2 Experimental Group 2 Normal Saline3 10 Quite obvious Large Control Group

By the postoperative observation for 1˜7 days, the inflammation andhealing of back wounds of the dogs in the HA fragment injectiontreatment groups 1, 2 were significantly better than those in the normalsaline treatment group.

Conclusion: The subcutaneous injection of HA fragment injectionseffectively controls the back wound inflammation of the dogs andpromotes the back wound healing of the dogs, indicating that the HAfragment injections have an anti-inflammatory effect on dogs as aspecies of large animals.

3.5. A Study of Controlling Oral Wound Inflammation in Cats.

15 pet cats with oral wound inflammation were selected from a pethospital, 5 cats were treated by local injection of 50 mg of HA fragmentInjection 1 (treatment group 1), 5 cats were treated by local injectionof 50 mg of HA fragment Injection 2 (treatment group 2), once a day for3 consecutive days, and the therapeutic effect was observed everymorning. The other 5 pet cats with oral wound inflammation from the pethospital were not treated as the control group.

Results: See Table 5.

TABLE 5 Results of Oral Wound Inflammation of the Cats Treated by HAFragment Injections. Number Redness and swelling of Redness and swellingof oral wound at Day 1 of oral wound at samples after treatment Day 3after treatment HA Fragment Injection 1 5 Obviously alleviated Almostdisappear Treatment Group 1 HA Fragment Injection 2 5 Obviouslyalleviated Almost disappear Treatment Group 2 Control Group 5 Notalleviated Not alleviated (untreated)

The observation at Day 1˜3 after the treatment with HA fragmentinjections showed that the local injection of HA fragment injection 1 orinjection 2 significantly controlled the redness and swelling of woundas the oral wound inflammation of the pet cats, and had a rapidanti-inflammatory effect on wound.

Conclusion: The HA fragment injections have a rapid anti-inflammatoryeffect on the oral wound inflammation of pet cats, and can significantlycontrol the redness, swelling and pain of wounds, indicating that the HAfragment injections have an anti-inflammatory effect on cats.

6. A Study of Controlling Inflammatory Cough in Cows.

Seven cows with inflammatory cough received by a veterinary hospitalwere treated by subcutaneous injection of 1 g of HA fragment Injection1, once a day for 3 consecutive days, and the therapeutic effect wasobserved every morning. Another 7 cows with inflammatory cough receivedby the veterinary hospital were not treated as the control group.

Results: See Table 6.

TABLE 6 Therapeutic Effect of HA Fragment Injection 1 on InflammatoryCough in Cows. Number of Cough at Day 1 Cough at Day 3 samples aftertreatment after treatment HA fragment 7 Obviously Almost disappearInjection 1 alleviated Treatment Group Control Group 7 Not alleviatedNot alleviated (untreated)

The observation at Day 1˜3 after treatment with HA fragment Injection 1showed that the inflammatory cough of the cows could be controlled at 3hours after subcutaneous injection of 1 g of HA fragment Injection 1,and it could be completely cured after 3 days of treatment.

Conclusion: The HA fragment injection is effective in treatinginflammatory cough in cows, indicating that the HA fragment injectionhas an anti-inflammatory effect on cows as a species of large animals.

3.7 Experiment of Controlling Arthritis in Horses.

Seven horses with arthritis received by a veterinary hospital weretreated by subcutaneous injection of 1 g of HA fragment Injection 1,once a day for 3 consecutive days, and the therapeutic effect wasobserved every morning.

Results: See Table 7.

TABLE 7 Therapeutic Effect of HA Fragment Injection 1 on Arthritis Painin Horses. Walking Walking abnormalities abnormalities caused by causedby Number arthritis pain arthritis pain of at Day 1 at Day 3 samplesafter treatment after treatment HA fragment 7 Begin to ObviouslyInjection 1 alleviate alleviated Treatment Group Control Group 7 Notalleviated Not alleviated (untreated)

At Day 1˜3 after treatment with HA fragment Injection 1, the observationon walking abnormalities caused by arthritis pain showed that thewalking abnormalities caused by arthritis pain in the horses weresignificantly alleviated at Day 1 after subcutaneous injection of 1 g ofHA fragment Injection 1, and the effect was more significant after 3days of treatment.

Conclusion: The HA fragment injection is effective in treating arthritispain of horses as large animals, indicating that the HA fragmentinjection has an anti-inflammatory effect on hoses as a species of largeanimals.

Summary on the researches in Embodiment 3: According to the researchresults in Embodiment 3, the research results of the function of LMW-HAfragments are completely different in mice and large animals, and thusit is predicted that the research results of the function of LMW-HAfragments in humans may be completely different from those in mice.

Embodiment 4

Research Background: According to the above researches in Embodiment 3,some researches on function of HA or its fragments using mice describedin previous references cannot represent the research results on functionof HA or its fragments in large animals and human, and the above LMW-HAfragment injections have “unexpected” different functions for human.

Objective: To study the anti-aging effect and safety of Injection 1 orB-HA injection (experiment group 1) containing protein residue ofrecombinant human hyaluronidase PH20 and Injection 2 (experimental group2) not containing protein residue of recombinant human hyaluronidasePH20 in the above Embodiment 1 on human facial cosmetology.

Methods: The HA fragment injection containing protein residue ofrecombinant human hyaluronidase PH20, i.e., HA fragment Injection 1(B-HA injection) in Embodiment 1, was used as the experimental group 1to carry out a clinical study among 98 subjects (with the mean age of62±18 years, 50 males and 48 females) with various diseases, subclinicalproblems or aging and aging-related diseases in current China'senvironment of air pollution, drinking water pollution and work stress;the HA fragment injection not containing protein residue of recombinanthuman hyaluronidase PH20, i.e., HA fragment Injection 2 in Embodiment 1,was used as the experimental group 2 to carry out a clinical study among100 subjects (with the mean age of 61±20 years, 49 males and 51 females)with various diseases or subclinical problems in current China'senvironment of air pollution, drinking water pollution and work stressor aging and aging-related diseases; they were injected with 100 mg (itrefers to 100 mg of HA fragments with a molecular weight distribution of10 KD-60 KD contained in the injection, the same below) via abdominalsubcutaneous fat layer, 1˜2 times a day for at least 7 consecutive days;

The cosmetic effects of the two injections (1 and 2) were compared byobserving the facial features or pictures of the subjects before andafter treatment (score: ineffective, undetermined, obviously effective),and the safety of the injection was evaluated by means of doctor'sinquiry about the subject's feeling, symptoms, physical examination andlaboratory tests, namely, whether there were side effects or not,including pain at the injection site, physical discomfort before andafter injection, mental state and physical strength, and changes inoriginal physical pain, original physical diseases and various originalsubclinical problems.

Results: See Table 8.

TABLE 8 Effect of the Two Injections (1 and 2) on Facial Cosmetology andAnti-aging. Number Obviously of Ineffective Undetermined effectivesamples Experimental 0 0  98  98 Group 1 (Injection 1) Experimental 0 0100 100 Group 2 (Injection 2) Note: Self-control was employed in thisstudy, which refers to the comparison between the subject’s currentstate (after treatment) and the state of a period before treatment.

FIG. 7 showed the comparison in the photos of some subjects before andafter treatment, as well as the therapeutic effect of the HA fragmentinjections on human facial cosmetology and facial aging; it alsoindicated that the HA fragment injections via abdominal subcutaneous fatlayer significantly reduced the thickness of subcutaneous fat tissue inhead, face, neck, shoulders, back, abdomen and upper and lower limbs.

TABLE 9 Effect of the Two Injections on Reducing Subcutaneous Fat.Number Obviously of Ineffective Undetermined effective samplesExperimental 6 18 74  98 Group 1 (Injection 1) Experimental 3 18 79 100Group 2 (Injection 2) Note: Self-control was employed in this study,which refers to the comparison between the subject’s current state(after treatment) the state of a period before treatment.

TABLE 10 Existence of Pain, Local Redness and Swelling, Local andSystemic Allergy After Deep Injection of the Two Injections viaAbdominal Subcutaneous Fat Layer. Number of No Undetermined Yes samplesExperimental  98 0 0  98 Group 1 (Injection 1) Experimental 100 0 0 100Group 2 (Injection 2)

In addition to the above cosmetic effects, safety and side effects, itwas also found that 9 cases felt comfortable and short period of sleepyafter injection via abdominal subcutaneous fat layer, the sleepinesslasted for 0.5˜1.5 h, wherein, 3 cases slept for 1.0 h after the firstinjection, and the energy and physical strength enhanced significantlyafter waking up; the other 6 cases did not feel sleepy any more afterinjection for twice.

TABLE 11 Side effect of Sleepiness After Both Injections. Number of NoUndetermined Yes samples Experimental 92 0 6  98 Group 1 (Injection 1)Experimental 97 0 3 100 Group 2 (Injection 2)

Unexpectedly, 185 of all the 198 subjects felt an enhancement in energyand physical strength at 30 min after injection via abdominalsubcutaneous fat layer, demonstrated by the desire to talk, wash dishes,cook, walk, plan to travel, work and no desire to rest, etc., whichlasted for 3˜4 days.

TABLE 12 Effect on Enhancing Energy and Physical Strength After BothInjections. Number of No Undetermined Obvious Samples Experimental 0 890  98 Group 1 (Injection 1) Experimental 0 5 95 100 Group 2 (Injection2) Note: Self-control was employed in this study, which refers to thecomparison between the subject’s current state (after treatment) thestate of a period before treatment.

77 of the 198 subjects (including 98 cases in the experimental group 1and 100 cases in the experimental group 2) suffered from different painsin shoulders, back, waist, legs and arm, and unexpectedly, they all feltthat the pains were relieved at 30 min after injection via abdominalsubcutaneous fat layer. Among them, 27 cases were additionally injectedwith 50 mg at the local painful site, and the pain began to relieve at 2min after the injection, which was equivalent to the effect of theprevious anesthesia treatment by local injection of lidocaine combinedwith prednisone complained by 12 cases.

TABLE 13 Analgesic Effects of the Two Injections (Note: mixed clinicaldata of Injection 1 and 2) Pain disappeared Pain disappeared afterinjection after injection treatment Pain began to treatment (15 × 100mg) relieve within (15 × 100 mg) without relapse Number 30 min after thewithout relapse within 6 months of first injection within 60 days ormore Samples Myofascitis in 17 20 16 21 waist, back, neck and upper andlower limbs Hyperosteogeny 20 26 16 30 and bone spur in waist, back andneck Old sports injury  8 14  6 12 Aging related or 12 14  8 14 unknownreasons Note: Self-control was employed in this study, which refers tothe comparison between the subject’s current state (after treatment) thestate of a period before treatment.

83 of the 198 subjects (including 98 cases in the experimental group 1and 100 cases in the experimental group 2) suffered from itching ofdifferent degrees, and unexpectedly, they all felt that the itching wasrelieved or disappeared at Day 1 after injection via abdominalsubcutaneous fat layer.

TABLE 14 Antipruritic Effect of the Two Injections (Note: mixed clincaldata of Injection 1 and 2). Itching did not Itching did not relapsewithin relapse within Itching began to 7 days after 60 days afterrelieve within injection injection 1 day after the treatment treatmentNumber of first injection (15 × 100 mg) (15 × 100 mg) samplesNeurodermatitis 15 13 12 16 Psoriasis 26 26 22 26 Senile Eczema 24 24 2024 Skin Scar 9 9 6 9 Inflammation and Hyperplasia Unknown 8 8 7 8reasons Note: Self-control was employed in this study, which refers tothe comparison between the subject’s current state (after treatment) andthe state of a period before treatment.

Conclusions:

-   1. Both HA fragment injections (Injection 1 and Injection 2)    containing and not containing protein residue of recombinant human    hyaluronidase have an effect on human facial cosmetology and facial    anti-aging.-   2. Both HA fragment injections (Injection 1 and Injection 2)    containing and not containing protein residue of recombinant human    hyaluronidase significantly reduce the thickness of subcutaneous fat    tissue in head, face, neck, shoulders, back, abdomen and upper and    lower limbs.-   3. Pain, local redness and swelling, as well as local and systemic    allergy are not observed after both HA fragment injections    (Injection 1 and Injection 2) containing and not containing protein    residue of recombinant human hyaluronidase were injected via    abdominal subcutaneous fat layer. It is convenient to use.-   4. After the first injection of both HA fragment injections    (Injection 1 and Injection 2) containing and not containing protein    residue of recombinant human hyaluronidase via abdominal    subcutaneous fat layer, the subjects feel comfortable and sleepy and    want to sleep, with an incidence rate less than 4.0%.-   5. Unexpectedly, both HA fragment injections (Injection 1 and    Injection 2) containing and not containing protein residue of    recombinant human hyaluronidase injected via abdominal subcutaneous    fat layer have an effect of significantly enhancing energy and    physical strength.-   6. Unexpectedly, both HA fragment injections (injection 1 and    Injection 2) containing and not containing protein residue of    recombinant human hyaluronidase injected via abdominal subcutaneous    fat layer have a rapid and significant analgesic (pain-relieving)    effect.-   7. Unexpectedly, both HA fragment injections (Injection 1 and    Injection 2) containing and not containing protein residue of    recombinant human hyaluronidase injected via abdominal subcutaneous    fat layer have an significant antipruritic effect.-   8. The above experimental results suggest that both HA fragment    injections of Injection 1 and Injection 2 in Embodiment 1 are the    equivalent HA fragment injections of the same kind, that is, 100 mg    of HA fragment injection with the same active ingredient of the same    amount.

Embodiment 5

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indications of myofascitis and old sports injuries of the HAfragment injections (Injection 1 and Injection 2).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of myofascitis and old sportsinjuries.

Results:

Rapid and effective treatment for myofascitis and old sports injuries:Myofascitis and old sports injuries are diseases related to sportsinjuries and senile degeneration, and the pathogenesis is not fullyclear. There are professional chiropractors specialized in treatingthese diseases in the United States, whereas there is still no curativemethod at present.

Among the 198 subjects, we analyzed 12 cases of myofascitis in waist,back, neck and upper and lower limbs (6 cases in the experimental group1 and 6 cases in the experimental group 2) and 14 cases of old sportsinjuries (6 cases in the experimental group 1 and 8 cases in theexperimental group 2), and the results were as shown in Table 15:

TABLE 15 Therapeutic Effect of HA Fragment Injections on Myofascitis andOld Sports Injuries Pain disappeared Pain disappeared after injectionafter injection treatment Pain began to treatment (15 × 100 mg) relievewithin (15 × 100 mg) without relapse 30 min after the without relapsewithin 12 months Number of first injection within 60 days or moresamples Myofascitis in 12 12  8 12 Waist, Back, Neck and Upper and LowerLimbs Old Sports 12 14 10 14 Injuries Note: Self-control was employed inthis study, which refers to the comparison between the subject’s currentstate (after treatment) the state of a period before treatment.

Among them, a 57-year-old male subject had suffered from myofascitis for3 years due to old sports injury for 17 years. It attacked when daysbecame cold every year, the pain was very afflicting, and certainmuscular movements may also cause the pain, and the range of activitywas limited for neck and back. The pain was relieved at 30 min afterinjection of 100 mg via abdominal subcutaneous fat layer, and then theeffect was better by additional deep injection of 100 mg at the localpain points of neck and back. After 15×100 mg of injections within 2months, the pain point and nodule on the back disappeared, and thethickness of neck and back muscles became thinner, without relapsewithin 12 months. The results showed that in addition to pain relief,the HA fragment injections finally solved the old sports injury andinflammatory lesions of myofascitis, realizing the purpose ofrecombination and regeneration of muscles and muscular fasciae.

Conclusion: The HA fragment injection via abdominal subcutaneous fatlayer or at pain point or diseased site effectively treats painfulmyofascitis as well as muscle and tendon injuries in waist, back, neckand shoulders.

Embodiment 6

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indications of bone spur and the protrusion of intervertebraldisc or diseases of lumbar spinal diseases and cervical vertebra of theHA fragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of bone spur and theprotrusion of intervertebral disc or lumbar spondylosis, back diseaseand cervical spondylosis.

Results:

HA fragment injections can achieve rapid and effective treatment of bonespur and the protrusion of intervertebral disc or lumbar spondylosis,back disease and cervical spondylosis. Bone spur and the protrusion ofintervertebral disc or lumbar spondylosis, back disease and cervicalspondylosis are diseases related to senile degeneration and sportsinjuries, the pathogenesis is not fully clear, and there is still nocurative method at present. Among the 198 subjects, we analyzed 30 casesof hyperosteogeny and bone spur in waist, back and neck (17 cases in theexperimental group 1 and 13 cases in the experimental group 2), and theresults were as shown in Table 16.

TABLE 16 Therapeutic Effect of HA Fragment Injections on the Protrusionof Intervertebral Disc or Lumbar Spondylosis, Back Disease and CervicalSpondylosis. Pain disappeared Pain disappeared after injection afterinjection treatment Pain began to treatment (15 × 100 mg) relieve within(15 × 100 mg) without relapse 30 min after the without relapse within 6months Number of first injection within 60 days or more samplesHyperosteogeny 26 30 23 30 and Bone Spur in Waist, Back and Neck Note:Self-control was employed in this study, which refers to the comparisonbetween the subject’s current state (after treatment) the state of aperiod before treatment.

Among them, a 57-year-old female subject suffered from hyperosteogenyand bone spur in waist, back and neck for 14 years. It attackedoccasionally with very afflicting pain, which may also by caused bycertain muscular movements. The range of activity was limited for neckand back, accompanied by the numbness of upper limbs and descendingworking ability. After injection of 100 mg via abdominal subcutaneousfat layer on Day 1, the subject complained that the pain was relieved bymore than 50%, and she could make dumplings and cook in the kitchen;after injection of 100 mg on Day 2, the subject complained that the painwas relieved by more than 80%, and after injection of 100 mg on Day 3,the subject complained that the pain was relieved by more than 95%.After 15×100 mg of injections within 2 months, it did not relapse anymore. The results showed that in addition to pain relief, the HAfragment injections finally started the recombination and regenerationof the inflammatory lesions of old sports injury.

Among them, another 57-year-old female subject suffered fromhyperosteogeny and bone spur in neck for 4 years. The range of activitywas limited for neck and back. It attacked occasionally with afflictingpain and was accompanied by the feeling of facial numbness, and certainmuscular movements may also cause the pain, accompanied by the numbnessof upper limbs and descending working ability. After injection of 100 mgvia abdominal subcutaneous fat layer on Day 1, the subject complainedthat the pain was relieved by more than 50% and after injection of 100mg on Day 2, the subject complained that the pain was relieved by morethan 80%. After 15×100 mg of injections within 2 months, it did notrelapse any more. The results showed that in addition to pain relief,the HA fragment injections also started the recombination andregeneration of the inflammatory lesions of old sports injury. Note: Allof the above subjects continued the treatment for more than 6 months towait for the result of relapse or not.

Among them, another 74-year-old male subject suffered from combinedlumbar spondylosis, back disease and cervical spondylosis for 20 years.The pain was afflicting and accompanied by movement disorders, and beprepared to hospitalize for operation. He was injected with the HAfragment injection via abdominal subcutaneous fat layer for 60×100 mgwithin 8 months, and he could walk and swim without pain, indicatingthat the conservative non-surgical treatment was successful; it alsosuggested that the HA fragment injection could finally cure theinflammatory lesions of old sports injury and promote the recombinationand regeneration of tissues.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer or at pain point or at diseased site is used for effectivelytreating the painful hyperosteogeny, bone spur, the protrusion ofintervertebral disc, and the lumbar spondylosis, back disease andcervical spondylosis.

Embodiment 7

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indications of vitreous opacity and vitreous detachment of theHA fragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of vitreous opacity andvitreous detachment.

Results:

HA fragment injection in rapid and effective treatment of vitreousopacity: Vitreous opacity is a senile degenerative disease, thepathogenesis of which is not fully clear, and there is still no curativemethod at present. Vitreous opacity also known as muscae volitantes willfurther develop into vitreous detachment that impairs the fundus andaffects the vision. Among the 198 subjects, we analyzed 19 cases ofvitreous opacity and vitreous detachment (11 cases in the experimentalgroup 1 and 8 cases in the experimental group 2), and the results wereas shown in Table 17:

TABLE 17 Therapeutic Effect of HA Fragment Injections on VitreousOpacity and Vitreous Detachment. The unilateral or Unilateral orbilateral spots of bilateral spots of muscae volitantes The visionmuscae volitantes relieved, dissipated was began to relieve, anddisappeared significantly dissipate and after injection ameliorateddisappear within treatment after 3 days after the (15 × 100 mg) 5 × 100mg Number of first injection without relapse injection Samples VitreousOpacity 19 19 9 19 (Muscae Volitantes) or Vitreous Detachment Note:Self-control was employed in this study, which refers to the comparisonbetween the subject’s current state (after treatment) the state of aperiod before treatment.

A 61-year-old female subject was treated by 100 mg of HA fragmentinjection via abdominal subcutaneous fat layer for the need of facialcosmetic treatment, and FIG. 6 showed the laboratory diagnosis of thesubject with vitreous opacity and vitreous detachment. After injection,it was occasionally found that the vision was ameliorated on Day 2, andthe spots of muscae volitantes for eyes became smaller on Day 3; she wasinjected with 15×100 mg every 3 days continuously. The vision keptameliorating (from 0.2 to 0.5) and the spots of muscae volitantes becamethinner and lighter continuously, without obvious relapse within 2months. The results showed that the HA fragment injection could rapidlyand effectively treat vitreous opacity (muscae volitantes) and vitreousdetachment surprisingly.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively treating muscae volitantes or vitreousopacity and the vitreous detachment and diminution of vision induced bythem.

Embodiment 8

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of wound scars with itching (chronic inflammation)and pain (inflammatory pain) of the HA fragment injections (HA fragmentInjections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of wound scars with itching(chronic inflammation) and pain (inflammatory pain).

Results:

HA fragment injections in the treatment of wound scars with itching(chronic inflammation) and pain (inflammatory pain): The small wounds ofpeople with scar diathesis can gradually form large scars and becomethickening and itching continuously. The therapeutic targets are torelieve the itching and reduce the scar thickening; the scars formedafter extensive burns without skin grafting continue to thicken withitching and chronic inflammation (FIG. 8 ), and the therapeutic targetsare to relieve the itching, eliminate the inflammation and reduce thescar thickening; surgical wounds mainly have redness, pain, inflammatoryreactive exudation and secondary infection, and therapeutic targets areto accelerate healing and reduce the scar formation. Among the 198subjects, we analyzed 14 cases of wound scars with itching (chronicinflammation) and pain (inflammatory pain) (5 cases in the experimentalgroup 1 and 9 cases in the experimental group 2), and the results wereas shown in Table 18:

TABLE 18 Therapeutic Effect of HA Fragment Injection on Wound Scars withItching (Chronic Inflammation) and Pain (Inflammatory Pain). The itchingThe scar became was thinner, the relieved, the inflammation pain wasaround scar relieved disappeared, the and the itching was The scarinflammation significantly became around relieved, and the thinner, andscar was pain was the itching relieved after significantly or paininjection of relieved after disappeared 1 × 100 mg injection of afterinjection Number of within 1 day 5 × 100 mg of 15 × 100 mg samples Scarsof Extensive 3 3 0 3 Burn Traumatic Scars 5 5 3 5 (ScarDiathesisantibiotics Scars of Surgical 6 6 5 5 Wound Note: Self-controlwas employed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

A 63-year-old female subject had extensive burns without skin grafting12 ago. She suffered from serious itching and difficulty in fallingasleep, and there were inflammations around the scars. At Day 3 after2×100 mg HA fragment injections, the scar became thinner, theinflammations around scar disappeared, the itching and the pain wassignificantly relieved; After 30×100 mg of HA injections continuously,the scar and the peripheral skin were significantly ameliorated,including that the scar became thinner, the inflammations disappeared,and the itching was relieved but did not completely disappear (FIG. 8 ).

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively treating extensive burns, traumatic scarsand scars of surgical wound accompanied by inflammations, itching andpain.

Embodiment 9

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of abdominal mucous membrane adhesion afteroperation or peritoneal dialysis of the HA fragment injections (HAfragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of abdominal mucous membraneadhesion after operation or peritoneal dialysis.

Results:

HA fragment injections in rapid and effective treatment of abdominalmucous membrane adhesion after operation or peritoneal dialysis:Surgical injury, abdominal dialysis, etc. can almost cause abdominalmucosal inflammations and adhesions, and can cause symptoms andpathological changes depending on the severity, including abdominalpain, abdominal tenderness, abdominal mass and intestinal obstruction.At present, there is still no feasible way to eradicate abdominaladhesions. Among the 198 subjects, we analyzed 10 cases of abdominalmucous membrane adhesion after operation or dialysis (5 cases in theexperimental group 1 and 5 cases in the experimental group 2), and theresults were as shown in Table 19:

TABLE 19 HA Fragment Injection in Rapid Treatment of Abdominal AdhesionsAfter Operation or Peritoneal Dialysis. Abdominal pain and Abdominalpain and Abdominal abdominal abdominal mass and tenderness weretenderness intestinal relieved, and the disappeared, and the obstructionenergy and physical energy and physical disappeared strength began tostrength significantly after turn better after turned better after 5 ×100 mg Number of 2 × 100 mg injections 5 × 100 mg injections injectionssamples Abdominal 7 7 7 7 Adhesions After Operation Abdominal 3 3 Notapplicable 3 Adhesions After Peritoneal Dialysis Note: Self-control wasemployed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

A 43-year-old female subject began to eat non-liquid food 1 month aftersubtotal gastrectomy and peripheral lymph node dissection, and thenabdominal mass and pain were found. She suffered from vomit aftereating, severe weight loss and poor health, and she was clinicallydiagnosed as intestinal obstruction induced by abdominal adhesions.After 5×100 mg of HA fragment injections via abdominal subcutaneous fatlayer, all the above symptoms and signs disappeared, intestinalobstruction did not occur after eating non-liquid food, and the mentalstate turned better significantly. The therapeutic results suggestedthat the HA fragment injection eventually eliminated the symptoms andsigns of abdominal adhesions maybe by reducing the inflammations inabdominal mucosa.

A 43-year-old female subject underwent oophorectomy began to receive 100mg of HA fragment injection via abdominal subcutaneous fat layer fromDay 1 preoperatively, once a day for 5 consecutive days. After theoperation, the mental state was obviously better than that after generaloperations. She could walk on the day of operation, she did not sufferfrom fever, she had bowel sounds and exhausted on the same day, and thepostoperative wound healing was obviously better that that after generaloperations.

A 53-year-old female subject underwent intestinal anastomosis began toreceive 100 mg of HA fragment injection via abdominal subcutaneous fatlayer from Day 1 preoperatively, once a day for 7 consecutive days.After the operation, the mental state was obviously better than thatafter general operations, she could walk slowly by holding the bed onthe day of operation, she did not suffer from fever, she had bowelsounds and exhausted on the same day, and the postoperative woundhealing was obviously better that that after general operations.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer or at diseased site can effectively treat pelvic mucous membraneadhesions after operation and peritoneal dialysis, effectively controlthe inflammatory symptoms caused by operation, obviously accelerate thepostoperative rehabilitation and reduce the postoperative pain.

Embodiment 10

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of endometriosis with dysmenorrhea or pelvic mucousmembrane adhesion of the HA fragment injections (HA fragment Injections1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of endometriosis withdysmenorrhea or pelvic mucous membrane adhesion.

Results:

HA fragment injections in the treatment of dysmenorrhea or pelvic mucousmembrane adhesion: Endometriosis is always accompanied by infertilityand dysmenorrhea; infertility and dysmenorrhea may be associated withthe inflammation and adhesion of pelvic mucous membrane, there is stillno effective method for treating dysmenorrhea and endometriosis orpelvic mucous membrane adhesion; among the 198 subjects, we analyzed 9cases of pelvic mucous membrane adhesion and dysmenorrhea (4 cases inthe experimental group 1 and 5 cases in the experimental group 2), andthe results were as shown in Table 20:

TABLE 20 HA Fragment Injection in Treating Endometriosis withDysmenorrhea or Pelvic Mucous Membrane Adhesion Dysmen- orrhea Dysmen-Dysmen- was orrhea orrhea significantly was almost relievedsignificantly disappeared within relieved after after 3 months 100 mg 2× 100 mg after injection injections 15 × 100 mg Number during the duringthe injections of menstruation menstruation continuously samplesEndometriosis 9 5 9 9 with dysmenorrhea Note: Self-control was employedin this study, which refers to the comparison between the subject’scurrent state (after treatment) the state of a period before treatment.

A 38-year-old female subject suffered from serious adenomyosis, theultrasonography showed that the uterus was obviously increased, anddysmenorrheal was very serious. She was injected with 100 mg of HAfragment via abdominal subcutaneous fat layer from the beginning ofmenstruation, once a day for 3 consecutive days, and the dysmenorrheawas obviously relieved.

A 41-year-old female subject suffered from serious adenomyosis, theultrasonography showed that the uterus was obviously increased, CA125was obviously higher than that of normal people, and the dysmenorrheawas serious. She was injected with 100 mg of HA fragment via abdominalsubcutaneous fat layer from the beginning of menstruation, once every 2days for 40 consecutive days, and the dysmenorrhea almost disappearedduring two menstruations, CA125 got normal, and ultrasonography showedthat the uterus began to reduce.

A 33-year-old female with infertility was found by examination to haveendometriosis and ovarian cyst; after the excision by minimally invasiveoperation, she began to receive 100 mg of HA fragment injection viaabdominal subcutaneous fat layer, once every 4 days for 6 consecutivemonths, and she was pregnant in the 7^(th) month.

The above results showed that the HA injections obviously relieveddysmenorrheal, and the HA injections reduced the inflammatory severityof endometriosis in pelvic cavity and the severity of referred paininduced by pelvic adhesion, indicating that the HA fragment injectionsmay also be used for treating infertility caused by poor peristalsis ofovarian ducts induced by endometriosis and pelvic adhesion.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively controlling dysmenorrhea and effectivelytreating endometriosis with dysmenorrhea and related diseases.

Embodiment 11

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indications of itching neurodermatitis, psoriasis, itchingsenile eczema, eczema herpeticum or pompholyx of the HA fragmentinjections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of itching neurodermatitis,psoriasis, itching senile eczema, eczema herpeticum or pompholyx.

Results:

HA fragment injections in the treatment of itching neurodermatitis,psoriasis, itching senile eczema, eczema herpeticum or pompholyx. Amongthe 198 subjects, we analyzed 29 cases of neurodermatitis, psoriasis,itching senile eczema, eczema herpeticum or pompholyx (14 cases in theexperimental group 1 and 15 cases in the experimental group 2), and theresults were as shown in Table 21:

TABLE 21 Therapeutic Effect of HA Fragment Injections on ItchingNeurodermatitis and Psoriasis. The itching The itching was was Theitching obviously obviously was relieved relieved, the relieved, the andthe skin skin skin inflammation inflammation inflammation began tovanished, and vanished, and vanish the skin lesions the skin lesionswithin 1 day became thinner disappeared after after after Number 1 × 100mg 10 × 100 mg 20 × 100 mg of injection injections injections samplesNeurodermatitis 6 6 6 6 Psoriasis 7 7 4 7 Note: Self-control wasemployed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

In Table 21, 1 subject of neurodermatitis (male, 57 years old) hadsuffered from neurodermatitis for 8 years, and he had bilateralsymmetrical multiple skin lesions, serious itching and localinflammatory skin lesions. The subject was dissatisfied with thetherapeutic effect of previous Stelara (anti-IL-12/23 antibody)injection. After 20×100 mg of HA fragment injections via abdominalsubcutaneous fat layer, the itching almost disappeared, the inflammatoryskin lesions almost recovered (FIG. 9 ), and the subject was satisfiedwith the effect.

In Table 21, 1 subject of psoriasis (male, 52 years old) had sufferedfrom psoriasis for 18 years, and he had multiple local inflammatory skinlesions and thickening of skin lesions in abdomen and the upper andlower limbs, accompanied by the itching symptom. The subject wasdissatisfied with the therapeutic effect of Stelara (anti-IL-12/23antibody) injection 2 years ago. After 10×100 mg of HA fragmentinjections via abdominal subcutaneous fat layer, the itching almostdisappeared, the thickened skin lesions obviously became thinner, theinflammatory skin lesions were obviously recovered (FIG. 10 ), and thesubject was satisfied with the effect.

In Table 21, 1 subject of psoriasis (female, 76 years old) had sufferedfrom psoriasis for 48 years, she had multiple local inflammatory skinlesions and thickening of skin lesions in abdomen and the upper andlower limbs, accompanied by the itching symptom, and cortical hormonehad not been used. After 20×100 mg of HA fragment injections viaabdominal subcutaneous fat layer, the itching disappeared, the skinlesions disappeared completely, and the subject was very satisfied withthe effect.

TABLE 22 Therapeutic Effect of HA Fragment Injections on Itching SenileEczema and Eczema Herpeticum or Pompholyx. The itching was relieved Theitching and the skin disappeared, inflammation and the began to vanishskin lesions almost within 1 day after disappeared after Number 1 × 100mg 10 × 100 mg of injection injections Samples Senile Eczema 13 13 13Pompholyx  3  3  3 Note: Self-control was employed in this study, whichrefers to the comparison between the subject’s current state (aftertreatment) the state of a period before treatment.

In Table 22, 1 subject of pompholyx (female, 33 years old) had sufferedfrom pompholyx for 9 years, and it attacked every summer. After 20×100mg of HA fragment injections via abdominal subcutaneous fat layer,pompholyx did not occur during injection and within 1 year afterinjection.

The results of Tables 21 and 22 showed that the HA fragment injectionsvia abdominal subcutaneous fat layer could treat itchingneurodermatitis, psoriasis, itching senile eczema, eczema herpeticum orpompholyx.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively treating itching neurodermatitis,psoriasis, itching senile eczema and eczema herpeticum or pompholyx.

Embodiment 12

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indications of senility-related diseases of the HA fragmentinjections (HA fragment Injections 1 and 2 in Embodiment 1), includingperiodontal inflammation, dental ulcer, guttural inflammation,conjunctival inflammation.

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of senility-related diseases,including periodontal inflammation, dental ulcer, guttural inflammation,conjunctival inflammation.

Results:

Senility is defined as the functional decline of various tissues andorgans, including the changes in skin and appearance and the decline ofphysical strength and energy. What is more outstanding is the frequentattacks of various common diseases, including senility-relatedperiodontal inflammation, dental ulcer, guttural inflammation andconjunctival inflammation. The HA fragment injections could rapidlytreat senility-related periodontal inflammation, dental ulcer, gutturalinflammation and conjunctival inflammation. The frequent attacks ofperiodontal inflammation, dental ulcer, guttural inflammation andconjunctival inflammation in old people were senility-related diseasesand were associated with the current China's environment of airpollution, drinking water pollution and greater work stress. Among the198 subjects, we analyzed 66 cases of periodontal inflammation, dentalulcer, guttural inflammation and conjunctival inflammation (36 cases inthe experimental group 1 and 30 cases in the experimental group 2), andthe results were as shown in Table 23:

TABLE 23 Therapeutic Effect of HA Fragment Injections on PeriodontalInflammation, Dental Ulcer, Guttural Inflammation and ConjunctivalInflammation. The Most of the inflammatory The inflammation inflammatorylesions were and pain began lesions were almost to vanish withineliminated eliminated 1 day after after after Number 1 × 100 mg 3 × 100mg 6 × 100 mg of injection injections injections samples Periodontal 6363 33 63 Inflammation Dental Ulcer  4  8  4  8 Inflammation 53 53 53 53of Pharynx and larynx Conjunctival 36 66 66 66 Inflammation Note:Self-control was employed in this study, which refers to the comparisonbetween the subject’s current state (after treatment) the state of aperiod before treatment.

The results of Table 23 showed that the HA fragment injections had morerapid therapeutic effect on senility-related periodontal inflammation,dental ulcer, guttural inflammation and conjunctival inflammation thanthe previous local spray of HA fragment injection.

Conclusion: 1. The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively treating senility-related periodontaldisease and dental ulcer, including the periodontal diseases and dentalulcer induced by chemotherapy and senility; 2. The HA fragmentinjections via abdominal subcutaneous fat layer are used for effectivelytreating senility-related guttural diseases, including the gutturaldiseases induced by chemotherapy and senility: 3. The HA fragmentinjections via abdominal subcutaneous fat layer are used for effectivelytreating senility-related conjunctival diseases, including theconjunctival diseases induced by chemotherapy and senility.

Embodiment 13

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of ankylosing spondylitis of the HA fragmentinjections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of ankylosing spondylitis.

Results: HA fragment injections could effectively treat ankylosingspondylitis.

Ankylosing spondylitis is a disease of inflammatory pain and seriousspinal deformation. Among the 198 subjects, we analyzed 3 cases of earlyankylosing spondylitis (2 cases in the experimental group 1 and 1 casein the experimental group 2), and the results were as shown in Table 24:

TABLE 24 Therapeutic Effect of HA Fragment Injections on AnkylosingSpondylitis. The pain Most of The pain began to the pain was was almostrelieve within eliminated eliminated 1 day after after after Number 1 ×100 mg 5 × 100 mg 10 × 100 mg of injection injections injections samplesAnkylosing 3 3 3 3 Spondylitis Note: Self-control was employed in thisstudy, which refers to the comparison between the subject’s currentstate (after treatment) the state of a period before treatment.

Early ankylosing spondylitis was a disease of inflammatory pain. Theanalgesic effect of the HA fragment injection treatment showed that theinflammation of ankylosing spondylitis was reduced or eliminated,suggesting that the serious spinal deformation induced by inflammationwould not develop continuously.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for effectively treating painful early ankylosingspondylitis.

Embodiment 14

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of recovery of physical strength and energy afterchemotherapy for tumor of the HA fragment injections (HA fragmentInjections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of recovery of physicalstrength and energy after chemotherapy for solid tumors.

Results.

HA fragment injection used for recovery of physical strength and energyafter chemotherapy for tumor. Among the 198 subjects, we analyzed 32cases requiring for the recovery of physical strength and energy afterchemotherapy for tumor (17 cases in the experimental group 1 and 15cases in the experimental group 2), and the results were as shown inTable 25:

TABLE 25 Therapeutic Effect of HA Fragment Injections on Recovery ofPhysical Strength and Energy and Alleviation of Chemotherapy-relatedMucous Complications (Including the Symptoms and Signs of Oral,Guttural, Conjunctival and Intestinal Mucous Complications) AfterChemotherapy for Solid Tumors. The related The physical The physicalmucous strength and strength and complications energy began energy wereto recover obviously obviously within 1 day recovered after alleviatedafter after 1 × 100 mg 5 × 100 mg 15 × 100 mg Number of injectioninjections injections samples After chemotherapy for 7 7 7 7 pancreaticcancer After operation and 5 5 5 5 chemotherapy for gastric cancer Afterradiotherapy and 11 11 11 11 chemotherapy for lung cancer Afteroperation and 1 1 1 1 chemotherapy for colorectal cancer Afterchemotherapy for 3 3 3 3 renal cancer After operation and 2 2 2 2chemotherapy for ovarian cancer After operation and 3 3 3 3 chemotherapyfor breast cancer Note: Self-control was employed in this study, whichrefers to the comparison subject’s current state (after treatment) thestate of a period before treatment.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer are used for recovering physical strength and energy andeffectively alleviating chemotherapy-related mucous complications afterchemotherapy for different tumors.

Embodiment 15

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of lung cancer of the HA fragment injections (HAfragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of lung cancer.

Results:

HA fragment injection used for treating late lung cancer: Among the 198subjects, we analyzed 7 cases with late lung cancer underwentchemotherapy combined with HA fragment injection (5 cases in theexperimental group 1 and 2 cases in the experimental group 2, once every3˜5 days for 12 consecutive months or till the death), and the resultswere as shown in Table 26:

TABLE 26 Therapeutic Effect of Chemotherapy Combined with HA FragmentInjections on Late Lung Cancer. Sizes of Lung Lung cancer or cancer orthe the metastatic Survival metastatic cancer of lung >12 months cancerof lung cancer did not for lung Number cancer was keep growing cancerafter of reduced and transfering treatment samples Chemotherapy 4 7 6 7Combined with HA Fragment Injection for Late Lung Cancer (ExperimentalGroups) Chemotherapy 2 0 1 7 for Late Lung Cancer (Control Group) Note:Self-control was employed in the experimental group of this study, whichrefers to the comparison between the subject’s current state (aftertreatment) the state of a period before treatment. The control group wasother cases of late lung cancer under chemotherapy only.

In Table 26, a 64-year-old female subject was diagnosed with lung cancerof the right lung, accompanied by lymphatic metastasis of the left lung,brain, bone and mediastinum, which was inoperable for excision, and shewas treated by radiotherapy and Tarceva chemotherapy. The subject wasvery weak after radiotherapy and chemotherapy, and started to receive100 mg of HA fragment injection via abdominal subcutaneous fat layer,once every 3˜5 days. The physical strength and energy of the subjectbegan to recover on Day 1 of injection. After 3 months of injection, theCT examination showed that the tumor and metastatic mass were reduced.After 12 months of injection, she still lived normally at home and didnot need hospitalization. The therapeutic results showed that the HAfragment injection could prevent the development and further metastasisof lung cancer and significantly prolong the survival.

In Table 26, a 78-year-old male subject was diagnosed with lung cancerof the right lung, accompanied by the lymphatic metastasis ofmediastinum, which was inoperable for excision, and the moleculartesting showed that the targeted drug chemotherapy would have nosignificant effect. The subject was still treated with chemical drugs,and started to receive 100 mg of HA fragment injection via abdominalsubcutaneous fat layer, once every 3 days for a total of 3 months. Thephysical strength and energy of the subject began to recover on Day 3 ofinjection. After 3 months of injection, the CT examination showed thatthe tumor and metastatic mass were reduced. After 12 months of injectioncontinuously, he still lived normally at home, the CT examination showedthat the reduced tumor and metastatic mass did not develop further, andit was unnecessary to hospitalize for further treatment. The therapeuticresults showed that the HA fragment injection could prevent thedevelopment and further metastasis of lung cancer.

HA fragment injections used for treating early lung cancer: Among the198 subjects, we analyzed 5 cases of early lung cancer underwentoperation combined with chemotherapy and HA fragment injection (3 casesin the experimental group 1 and 2 cases in the experimental group 2,once every 3˜5 days for 3 consecutive months from 1 weekpreoperatively), and the results were as shown in Table 27:

TABLE 27 Effect of Operation Combined with Chemotherapy and HA FragmentInjections on Preventing Relapse of Early Lung Cancer. The physicalstrength recovered to normal and maintained Lung for cancer Survival >12months did not >12 months after relapse for lung Number operation forwithin cancer after of lung cancer 12 months treatment samples OperationCombined 5 5 5 5 with B-HAHA fragment Injection for Early Lung Cancer(Experimental Groups) Operation Combined 0 2 1 5 with Chemotherapy forEarly Lung Cancer (Control Groupantibiotics Note: Self-control wasemployed in the experimental group of this study, which refers to thecomparison between the subject’s current state (after treatment) thestate of a period before treatment. The control group was other casestreated by operation combined with chemotherapy only.

Conclusion: 1. The experimental results show that the HA fragmentinjections can prevent the development and further metastasis of latelung cancer; 2. The experimental results also show that the HA fragmentinjections could prevent the relapse of early lung cancer afteroperation.

Embodiment 16

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of pancreatic cancer of the HA fragment injections(HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of pancreatic cancer.

Results:

Pancreatic cancer as a quite malignant tumor develops rapidly aftermetastasis, and it will lead to death within 90 days to the fastest.Foreign researches have shown that inflammation and tumor-relatedinflammatory cells may be the important stimulatives for the developmentand metastasis of pancreatic cancer.

HA fragment injections used for treating late pancreatic cancer: Amongthe 198 subjects, we analyzed 5 cases of late pancreatic cancerunderwent operation combined with chemotherapy and HA fragmentinjections (3 cases in the experimental group 1 and 2 cases in theexperimental group 2, once every 3˜5 days for 12 consecutive months ortill the death from 2 weeks preoperatively), and the results were asshown in Table 28:

TABLE 28 Therapeutic Effect of Chemotherapy Combined with B-HAHAFragment Injections on Late Pancreatic Cancer. Sizes of late Latepancreatic pancreatic cancer or the cancer or the metastatic metastaticmass of mass late pancreatic of late cancer did not Survival pancreaticcontinue >12 months Number cancer was growing and after of reducedtransfering treatment samples Chemotherapy 2 4 4 5 Combined with HAFragment Injection for Late Pancreatic Cancer (Experimental Groups)Chemotherapy 0 0 0 5 for Late Pancreatic Cancer (ControlGroupantibiotics Note: Self-control was employed in the experimentalgroup of this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment. The control group was other cases treated by chemotherapy.

In Table 28, a subject (male, 46 years old) after chemotherapy forpancreatic head cancer after chemotherapy was found that the pancreatichead cancer had transferred to liver due to jaundice, the bile duct wasblocked to result in jaundice, the jaundice was solved by placing astent through operation and nasobiliary drainage. He receivedchemotherapy monthly and was treated by traditional Chinese medicine atthe same time. After 5×100 mg of HA fragment injections via abdominalsubcutaneous fat layer, the physical strength and energy recovered;after 20×100 mg of HA fragment injections via abdominal subcutaneous fatlayer, the ultrasonography showed that the pancreatic head cancer andthe metastatic mass to liver began to reduce, and the subject workednormally for more than 90 days. The above results suggested that the HAfragment injection could promote the recovery of physical strength andenergy after chemotherapy for solid tumors and might prevent thedevelopment and further metastasis of pancreatic head cancer byinhibiting inflammations. According to a foreign report (HalozymeReceives Orphan Drug Designation For PEGylated Recombinant HumanHyaluronidase PH20 For Pancreatic Cancer at:http://www.halozyme.com/Investors/News-Releases/News-Release-Details/2014/Halozyme-Receives-Orphan-Drug-Designation-For-PEGylated-Recombinant-Human-Hyaluronidase-PH20˜For-Pancreatic-Cancer/default.aspx#sthash.aLjvxToQ.dpuf),long-acting hyaluronidase PEGPH20 combined with chemotherapy effectivelyprolongs the survival of patients with pancreatic cancer, and theexperimental results indicate that the mechanism of action might be thatthe LMW-HA fragment injection similar to the invention is produced usingHA in human body.

Conclusion: The experimental results show that the HA fragmentinjections can prevent the development and further metastasis of latepancreatic cancer and obviously prolong the survival.

Embodiment 17

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of gastric cancer of the HA fragment injections (HAfragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of gastric cancer.

Results:

HA fragment injections used for treating late gastric cancer: Among the198 subjects, we analyzed 4 cases of late gastric cancer underwentoperation combined with chemotherapy and HA fragment injections (3 casesin the experimental group 1 and 1 case in the experimental group 2, onceevery 1˜5 days for >12 consecutive months), and the results were asshown in Table 29:

TABLE 29 Therapeutic Effect of Chemotherapy Combined with B-HAHAFragment Injections on Late Gastric Cancer. Late gastric cancer Sizes ofor the late gastric metastatic cancer or mass of the late gastricmetastatic cancer mass of did not Survival late gastric continue >12months Number cancer was growing and after of reduced transferingtreatment samples Chemotherapy 2 5 5 5 Combined with HA FragmentInjection for Late Gastric Cancer (Experimental Groups) Chemotherapy for0 2 2 5 Late Gastric Cancer (Control Groupantibiotics Note: Self-controlwas employed in the experimental group of this study, which refers tothe comparison between the subject’s current state (after treatment) thestate of a period before treatment. The control group was other casestreated by chemotherapy.

In Table 29, a subject (male, 62 years old) after chemotherapy forgastric cancer had multiple metastasis of gastric cancer, and themetastatic mass in brain compressed the respiratory center to causedyspnea, and he was rescued in the ICU. He was treated by 100 mg of HAfragment injection via abdominal subcutaneous fat layer, once a day for20 consecutive days. At Day 10 after HA fragment injection, the subjectbreathed smoothly with the physical strength and energy recovered, andhe was discharged to go home for rehabilitation. After discharge, thesubject was injected with 100 mg of HA fragment injection via abdominalsubcutaneous fat layer at home, once every 3˜5 days for 12 consecutivemonths or more.

Conclusion: The experimental results show that the HA fragmentinjections can prevent the development and further metastasis of lategastric cancer and obviously prolong the survival.

Embodiment 18

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of ovarian cancer of the HA fragment injections (HAfragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of ovarian cancer.

Results: HA fragment injection used for treating late ovarian cancer:Among the 198 subjects, we analyzed 5 cases of late ovarian cancerunderwent chemotherapy combined with HA fragment injection (2 cases inthe experimental group 1 and 3 case in the experimental group 2, onceevery 3 days for 3 consecutive months), and the results were as shown inTable 30:

TABLE 30 Therapeutic Effect of Chemotherapy Combined with B-HAHAFragment Injections on Late Ovarian Cancer. Late ovarian cancer or themetastatic CA125 CA125 mass of late was was ovarian cancer slightlysignificantly did not continue lowered lowered Number growing and afterafter of transfering treatment treatment samples Chemotherapy 5 0 5 5Combined with HA Fragment Injection for Late Ovarian Cancer(Experimental Groups) Chemotherapy for 1 3 1 5 Late Ovarian Cancer(Control Group) Note: Self-control was employed in the experimentalgroup of this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment. The control group was other cases treated by chemotherapy.

In Table 30, a subject (female, 62 years old) after operation andchemotherapy for ovarian cancer was extremely weak due to thechemotherapy and radiotherapy for lymphatic metastasis, and she had tostay in bed all day. She was injected with 20×100 mg of HA fragmentinjections via abdominal subcutaneous fat layer, the physical strengthand energy began to recover on Day 2 after 1×100 mg; at the end of20×100 mg injections, CA125 was surprisingly reduced to 50 U/ml from 500U/ml before treatment, and the tumor and metastatic mass were reduced,indicating that the HA fragment injection could promote the recovery ofphysical strength and energy after chemotherapy for treating ovariancancer and might prevent the development and further metastasis ofovarian cancer by inhibiting inflammations.

Conclusion: The experimental results show that the HA fragmentinjections can lower CA125 as a cancer indicator for late ovarian cancerand control the continuous growth and metastasis of late ovarian canceror the metastatic mass of late ovarian cancer.

Embodiment 19

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of oral cancer with difficulty in eating of the HAfragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of oral cancer with difficultyin eating.

Results: HA fragment injections used for treating oral cancer: Among the198 subjects, we analyzed 10 cases of oral cancer with obviousdifficulty in eating treated by HA fragment injection (6 cases in theexperimental group 1 and 4 cases in the experimental group 2, once a dayfor 15 injections consecutively for those in the experimental group inthe Table, and injection of normal saline+oral administration ofantibiotics metronidazole for those in the control group), and theresults were as shown in Table 31:

TABLE 31 Therapeutic Effect of HA Fragment Injections on Oral Cancerwith Obvious Difficulty in Eating The mass of oral The difficulty incancer and the eating induced induced The mass of oral by the mass ofinflammation cancer did not oral cancer became smaller become smalleralmost Number of and vanished and vanished disappeared samples HAFragment 5 0 5 5 Injection + Oral Administration of AntibioticsMetronidazole (Experimental Groups) Injection of 2 3 1 5 normal saline +Oral Administration of Antibiotics Metronidazole (ControlGroupantibiotics

Conclusion: The experimental results show that the HA fragmentinjections can be used for treating oral cancer with obvious difficultyin eating.

Embodiment 20

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of postoperative relapse of meningioma of the HAfragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of postoperative relapse ofmeningioma.

Results:

HA fragment injection used for treating postoperative relapse ofmeningioma: Among the 198 subjects, we analyzed 5 cases of postoperativerelapse of meningioma treated by HA fragment injection (3 cases in theexperimental group 1 and 2 cases in the experimental group 2, once every3˜5 day for 12 consecutive months), and the results were as shown inTable 32:

TABLE 32 Therapeutic Effect of HA Fragment Injections on PostoperativeRelapse of Meningioma. The enhanced MRI showed The headache The headachethat the symptom and symptom and meningioma number of number of residuleattacks were headaches did not obviously were continue to Numberrelieved or aggravated thicken or of disappeared or increased relapsesamples HA Fragment 5 0 5 5 Injection Experimental Groups Blank Control0 5 0 5 Group Note: Self-control was employed in the experimental groupof this study, which refers to the comparison between the subject’scurrent state (after treatment) the state of a period before treatment.The control group was other cases given blank treatment.

In Table 32, a female subject (62 years old) was diagnosed withmeningioma by enhanced MRI for frequent headache in 2007, and theheadache disappeared after operation in the same year. From 2015, theheadache attacked frequently, and she was diagnosed with meningiomaresidual or relapse by enhanced MRI, namely that the meninges wasobviously thickened. After 100 mg of HA fragment injection via abdominalsubcutaneous fat layer for 15 times, the attacks of headache wereobviously reduced. The subject continued the injection once every 5days, 100 mg every time for about 60 times within the following 12months. The results of enhanced MRI showed that the focus of previousmeningioma residual or relapse was reduced from 18.3×12.7×112.2 mm to16.3×7.9×13.2 mm (FIG. 11 ), the focus was reduced instead of growingcontinuously, and finally the subject did not suffer from the attack ofheadache any more. The above results showed that the HA fragmentinjection could be used for treating meningioma or the postoperativerelapse of meningioma.

Conclusion: The experimental results show that the HA fragmentinjections can control the postoperative relapse of meningioma.

Embodiment 21

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of functional recovery after cerebral embolism ofthe HA fragment injections (HA fragment Injections 1 and 2 in Embodiment1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of functional recovery aftercerebral embolism.

Result 1: HA fragment injection used for functional recovery aftercerebral embolism: Among the 198 subjects, we analyzed 3 cases requiringfor functional recovery for the stiffness of upper limbs after cerebralembolism (3 cases in the experimental group 1, 1 injection every 2 daysfor 10 times consecutively), and the results were as shown in Table 33:

TABLE 33 Effect of HA Fragment Injections on Functional Recovery for theStiffness of Upper Limbs After Cerebral Embolism The stiffness Thestiffness The of upper of upper stiffness limbs and limbs and of upperthe failure the failure limbs and in relaxing in relaxing the failurefingers fingers in relaxing began to turn obviously fingers betterwithin turned further turned 1 day after better after better afterNumber 1 × 100 mg 5 × 100 mg 10 × 100 mg of injection injectionsinjections samples HA Fragment 3 3 3 3 Injection Note: Self-control wasemployed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

In Table 33, 1 male subject (79 years old) had suffered from cerebralembolism for 3 years, and he had the feel of cold on the whole body,stiffness of upper limbs and failure in relaxing fingers. After 10×100mg HA fragment injections via abdominal subcutaneous fat layer, thesymptoms and signs of feel of cold on the whole body, stiffness of upperlimbs and failure in relaxing fingers obviously turned better, and herequired to receive the HA fragment injection continuously.

In Table 33, 1 male subject (82 years old) had suffered from cerebralembolism for 8 years, and he had the stiffness of upper limbs, failurein relaxing fingers and pain in fingers. After 10×100 mg HA fragmentinjections via abdominal subcutaneous fat layer, the symptoms and signsof stiffness of upper limbs and failure in relaxing fingers obviouslyturned better.

In Table 33, 1 male subject (56 years old) had suffered from cerebralembolism for 1 year, and he had the stiffness of upper limbs, failure inrelaxing fingers and numbness in fingers. After 10×100 mg HA fragmentinjections via abdominal subcutaneous fat layer, the symptoms and signsof stiffness of upper limbs, failure in relaxing fingers and numbness infingers obviously turned better.

V. Result 2: HA fragment injections used for functional recovery aftercerebral embolism: Among the 198 subjects, we analyzed 4 cases requiringfor functional recovery for the weakness of upper limbs after cerebralembolism (1 case in the experimental group 1 and 3 cases in theexperimental group 2), and the results were as shown in Table 34:

TABLE 34 Effect of HA Fragment Injections on Functional Recovery AfterCerebral Embolism The weakness in The weakness in half body half bodyslightly turned obviously turned better and it was better and it was Theenergy and a little stronger strong to walk or physical strength to walkor the the vision were improved vision began to obviously turned within1 day turn better after better after after 1 × 100 mg 5 × 100 mg 10 ×100 mg Number of injection injections injections samples Weakness in 3 33 3 Half Body, Feebleness in Walking Diminution of 1 1 1 1 Vision Note:Self-control was employed in this study, which refers to the comparisonbetween the subject’s current state (after treatment) the state of aperiod before treatment.

In Table 34, 1 male subject (67 years old) had suffered from cerebralembolism for 30 days, and he was weak in half body and feeble inwalking. After 10×100 mg HA fragment injections via abdominalsubcutaneous fat layer, the weakness in half body obviously turnedbetter, and he was strong to walk, indicating that the HA fragmentinjection could be used for the functional recovery after cerebralembolism.

In Table 34, 1 male subject (59 years old) had suffered from cerebralembolism for 4 days, and he could only see the objects within 20 cm dueto the impaired vision. After 10×100 mg HA fragment injections viaabdominal subcutaneous fat layer, the vision obviously turned better,and he could see the objects within 40 cm, indicating that the HAfragment injection could be used for the functional recovery aftercerebral embolism.

Conclusion: The HA fragment injections via abdominal subcutaneous fatlayer can effectively promote the functional recovery after cerebralembolism.

Embodiment 22

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of angina of the HA fragment injections (HA fragmentInjections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of angina.

HA fragment injections used for treating angina induced bycardiovascular diseases: Coronary heart disease, complications afterintravascular stent and other cardiovascular diseases are essentiallychronic inflammatory diseases, and the effective methods for preventionand treatment are in urgent need. Among the 198 subjects treated by HAfragment injections, we analyzed 6 cases of angina (3 cases in theexperimental group 1 and 3 cases in the experimental group 2, once a dayfor 20 injections consecutively), and the results were as shown in Table35:

TABLE 35 Therapeutic Effect of HA Fragment Injections on Angina Inducedby Cardiovascular Diseases. The energy The attacks and physical ofangina Angina strength were did not were improved obviously relapsewithin 1 day reduced within Number after after 60 days after of 1injection 10 injections 20 injections samples Coronary Heart 3 3 3 3Diseases Complications 3 3 3 3 after intravascular stent Note:Self-control was employed in this study, which refers to the comparisonbetween the subject’s current state (after treatment) the state of aperiod before treatment.

Conclusion: The results of Table 35 show that the HA fragment injectionvia abdominal subcutaneous fat layer can effectively treat the anginainduced by cardiovascular diseases.

Embodiment 23

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of painful gout during attack of the HA fragmentinjections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indications of painful gout duringattack.

HA fragment injections used for treating gout: Among the 198 subjectstreated by HA fragment injections, we analyzed 5 cases of painful goutduring attack (3 cases in the experimental group 1 and 2 cases in theexperimental group 2, once a day for 5 times consecutively), and theresults were as shown in Table 36:

TABLE 36 Therapeutic Effect of HA Fragment Injections on Painful GoutDuring Attack. The symptom The sign of The of red and pain and thesymptom swelling sign of red of pain was toes was and swelling obviouslyobviously toes almost relieved relieved disappeared Number after afterafter of 1 injection 1 injection 5 injections samples HA Fragment 5 5 55 Injection Note: Self-control was employed in this study, which refersto the comparison between the subject’s current state (after treatment)the state of a period before treatment.

Conclusion: The results of Table 36 showed that the HA fragmentinjection via abdominal subcutaneous fat layer can effectively treatpainful gout during attack.

Embodiment 24

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of arthritis never treated by cortical hormone ofthe HA fragment injections (HA fragment Injections 1 and 2 in Embodiment1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of arthritis never treated bycortical hormone.

HA fragment injections used for treating arthritis never treated bycortical hormone: Among the 198 subjects treated by HA fragmentinjections, we analyzed 6 cases of arthritis never treated by corticalhormone (3 cases in the experimental group 1 and 2 cases in theexperimental group 2, once a day for 15 times consecutively), and theresults were as shown in Table 37:

TABLE 37 Therapeutic Effect of HA Fragment Injections on Arthritis NeverTreated by Cortical Hormone. The symptom of The sign of arthralgia andredness and the sign of The symptom swelling redness and of arthralgiaof joint swelling of joint began to began to were obviously Numberrelieve after relieve after relieved after of 1 injection 1 injection 15injections samples Osteoarthritis 3 0 3 3 Rheumatic 2 0 3 3 ArthritisNote: Self-control was employed in this study, which refers to thecomparison between the subject’s current state (after treatment) thestate of a period before treatment.

HA fragment injections used for treating arthritis previously treated bycortical hormone: Among the 198 subjects treated by HA fragmentinjections, we analyzed 4 cases of arthritis previously treated bycortical hormone (2 cases in the experimental group 1 and 2 cases in theexperimental group 2, once a day for 15 times consecutively), and theresults were as shown in Table 38:

TABLE 38 Therapeutic Effect of HA Fragment Injection on ArthritisPreviously Treated by Cortical Hormone. Compared with that Compared withCompared before those before with that treatment, treatment, the beforethe sign of symptom of treatment, redness and arthralgia and the thesymptom swelling sign of redness and of arthralgia of joint swelling ofjoint began to began to were obviously Number relieve after relieveafter relieved after of 1 injection 1 injection 15 injections samplesOsteoarthritis 0 0 1 2 Rheumatic 0 0 0 2 Arthritis Note: Self-controlwas employed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

Conclusion: 1. The HA fragment injection via abdominal subcutaneous fatlayer can effectively treat arthritis never treated by cortical hormone;2. The HA fragment injection via abdominal subcutaneous fat layer had notherapeutic effect on arthritis previously treated by cortical hormone.

Embodiment 25

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of herpes zoster with serious pain of the HAfragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of herpes zoster with seriouspain.

HA fragment injections used for treating herpes zoster with seriouspain: Among the 198 subjects treated by HA fragment injections, weanalyzed 5 cases of herpes zoster with serious pain (2 cases in theexperimental group 1 and 2 cases in the experimental group 3, once a dayfor 10 times consecutively), and the results were as shown in Table 39:

TABLE 39 Therapeutic Effect of HA Fragment Injections on Herpes Zosterwith Serious Pain. The symptom The symptom of pain of pain was almostobviously disappeared Number relieved after after of 1 injection 10injections samples HA Fragment 5 5 5 Injection Note: 1. Self-control wasemployed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment; 2. In the untreated blank control group, the pain wasaggravated in 2 cases and the pain was not relieved in 3 cases within 10days.

Conclusion: The results of Table 39 show that the HA fragment injectionsvia abdominal subcutaneous fat layer can effectively treat herpes zosterwith serious pain.

Embodiment 26

Objective: To further analyze the changes in the diseases andsubclinical problems of the 198 subjects before and after treatment withHA fragment injections in “Embodiment 4”, and to identify the newclinical indication of acute pancreatitis with serious pain of the HAfragment injections (HA fragment Injections 1 and 2 in Embodiment 1).

Methods: The changes in the diseases and subclinical problems of the 198subjects before and after treatment with HA fragment injections in“Embodiment 4” were further analyzed, including medical history,symptoms, signs and laboratory results before injection, and the changesin symptoms, signs and laboratory results after the HA fragmentinjections, so as to identify the therapeutic effect of the HA fragmentinjections on the new clinical indication of acute pancreatitis withserious pain.

HA fragment injections used for acute pancreatitis with serious pain:Among the 198 subjects treated by HA fragment injections, we analyzed 6cases of acute pancreatitis with serious pain (3 cases in theexperimental group 1 and 3 cases in the experimental group 2, in thetable below, the experimental groups were injected with HA fragmentinjection+antibiotics for once a day, and the control group was injectedwith antibiotics for once a day, for 10 injections consecutively), andthe results were as shown in Table 40:

TABLE 40 Therapeutic Effect of HA Fragment Injections on AcutePancreatitis. The symptom The of pain was symptom The obviously of painsymptom relieved disappeared of pain and the and the started to subjectate subject relieve liquid food ate normal on after after food afterNumber 1 day of 3 days of 10 days of of injection injection injectionsamples HA Fragment 3 3 3 3 Injection + Antibiotics (ExperimentalGroups) Antibiotics 0 0 0 3 (Control Group) Note: Self-control wasemployed in the experimental group of this study, which refers to thecomparison between the subject’s current state (after treatment) thestate of a period before treatment.

Conclusion: The results of Table 40 show that the HA fragment injectionvia abdominal subcutaneous fat layer can effectively treat acutepancreatitis with serious pain.

Embodiment 27

Objective: To compare the studies on subcutaneous injection of LMW-HAfragments produced by different methods on dogs and human subjects.

Methods: In Embodiment 1 of the invention, the HMW-HA raw material wasdirectly digested using human recombinant hyaluronidase to produce HAfragment Injection 1 and Injection 2 (with a molecular weightdistribution of 10˜60 KD), and the injection deep into abdominalsubcutaneous fat layer did not cause pain in the above embodiment.

In the previous research achievement (Application No.: 201410153593.5,Publication No.: CN105018547A) of the applicant, it is also recordedthat HMW-HA raw material was firstly baked and heated to reduce themolecular weight of HMW-HA raw material, and then HMW-HA was digested byrecombinant human hyaluronidase to produce the LMW-HA fragments.

In this embodiment, the step of baking HMW-HA raw material is added inreference to the above method. The baking aims to reduce the molecularweight of HMW-HA raw material and reduce the viscosity after dissolutionusing the physical method. Besides, owing to the reduction in themolecular weight of HMW-HA raw material after baking, the amount ofrecombinant human hyaluronidase used for subsequent digestion can befurther reduced to greatly lower the cost.

In implementation, except the baking step, the rest of steps are thesame as those in Embodiment 1. Specifically, the process ofimplementation can follow the specific steps below:

Use an enzyme digestion and blending tank with a working volume of 25 Lfor cleaning and sterilization in-place, and prepare injection-grade HAraw material with a molecular weight distribution of 800˜1,200 KD (thesame as those in Embodiment 1); Treat the injection-grade HA rawmaterial with a molecular weight distribution of 800˜1,200 KD as below:Bake at 120° C. for 1˜2 h (treatment 1) or bake at 105° C. for 5˜6 h(treatment 2);

Add injection-grade pure water into the injection-grade HA raw materialwith a molecular weight distribution of 800˜1,200 KD after baking by onetime or several times, then add 80˜90 mM of sodium chloride, 1 mM ofmagnesium ions and 4,000˜5,000 units of recombinant human hyaluronidase(the preparing method is the same as that in Embodiment 1) per 1 g ofHMW-HA raw material in order, mix them completely, and react at 37° C.for 5˜6 h until that the molecular weight of HA fragments reaches 10KD-60 KD. Add 35˜45 mM of sodium chloride to regulate the osmoticpressure to 280˜300 mOsm/L, then heat it at 84˜95° C. for 30˜60 min(thermal inactivation of recombinant human hyaluronidase, partialbacteria inactivation and virus inactivation for reducing the pH), andthen filter bacteria at 0.22 μm.

The HA fragment injection was prepared after filtration, comprising:

Injection 3 (by treatment 1, including the step of baking at 120° C.):

Each 2-4 ml contained 100 mg of HA fragments with a molecular weightdistribution of 10 KD˜60 KD and <20 μg of protein residue of inactivatedrecombinant human hyaluronidase PH20 without enzyme activity(endotoxin<0.5 IU/ml, sterile, virus-free), the final concentration ofsodium chloride was 115˜125 mM, and the final concentration of magnesiumions was 1 mM.

Injection 4 (by treatment 2, including the step of baking at 105° C.):

Each 2˜4 ml contained 100 mg of HA fragments with a molecular weightdistribution of 10 KD˜60 KD and <20 μg of protein residue of inactivatedrecombinant human hyaluronidase PH20 (endotoxin<0.5 IU/ml, sterile,virus-free), the final concentration of sodium chloride was 115˜125 mM,and the final concentration of magnesium ions was 1 mM.

The LMW-HA fragment Injection 3 and Injection 4 as well as Injection 1in the above embodiment produced by different methods were used forsubcutaneous injection on dogs and human, and it was found thatInjection 3 induced the side effect of pain in injection. The pain indogs was demonstrated by the fierce flounder at the beginning ofinjection and in the process of injection, and the pain in subjects wasdemonstrated by complaining about the pain to the injector and requiringstopping the injection immediately.

Results: See Table 41.

TABLE 41 Results of Study on Subcutaneous Injection of LMW-HA FragmentsProduced by Different Methods in Causing Pain for Dogs and Human.Production Method of LMW-HA Number of Manner of Fragment InjectionSamples Injection Dog Subject Baking at 120° C. for 3 Subcutaneous Thedog The subject felt 1~2 h + Subsequent floundered painful as soon asEnzyme Digestion by fiercely at the the injection began RecombinantHuman beginning of and required Hyaluronidase injection and stopping the(Injection 3) in the process injection of injection immediately Bakingat 105° C. for 3 Subcutaneous The dog was The subject did not 5~6 h +Subsequent quiet at the feel painful and Enzyme Digestion by beginningof kept quiet at the Recombinant Human injection and beginning ofHyaluronidase in the process injection and in the (Injection 4) ofinjection process of injection Direct Enzyme 3 Subcutaneous The dog wasThe subject did not Digestion by quiet at the feel painful andRecombinant Human beginning of kept quiet at the Hyaluronidase injectionand beginning of Without Baking in in the process injection and in theAdvance (Injection 1) of injection process of injection

Conclusion: 1. Unexpectedly, the subcutaneous injection of LMW-HAfragments produced by baking at 120° C. causes serious pain for dogs andsubjects, and it can't be used for injection; 2. Unexpectedly, thesubcutaneous injection of LMW-HA fragments produced by baking at 105° C.does not cause pain for dogs and subjects, and it can be used forinjection (the injection also has a therapeutic effect on the abovediseases or symptoms in subjects only with enough sample number in orderto achieve statistical significance, and the specific results are notlisted here. Besides, in consideration to the production cost, the HAfragment injection produced by this production method is applicable fordeveloping into products for experimental animals or for veterinaryuse); 3. The subcutaneous injection of LMW-HA fragments produced withoutbaking and directly by enzyme digestion using recombinant humanhyaluronidase does not cause pain for dogs and subjects, and it can beused for injection.

Embodiment 28

Objective: To explore the possibility for activation and commercializedproduction of LMW-HA fragments with the therapeutic effect in the aboveembodiments by the digestion using other types of hyaluronidase.

Methods: (1) Study the production methods of multiple hyaluronidases andthe results of producing LMW-HA fragments by enzyme digestion of HMW-HAfound in the existing references; (2) Produce different hyaluronidasesindependently or cooperatively, and verify the feasibility in enzymedigestion and activation of HMW-HA; (3) Study what kind of hyaluronidasecan digest and activate HMW-HA to the corresponding molecular weight?Which kind of end structure of enzyme digestion can activate LMW-HA tohave the therapeutic effect of the HA fragment injections shown in theabove embodiments? Which kind of hyaluronidase residual will not causeallergic reaction in human?

Results and Discussions: By combining with the production methods ofmultiple hyaluronidases and the methods for enzyme digestion of HMW-HA,the research results are respectively reported as below:

-   1. In addition to neutral human acrosome hyaluronidase PH20 (or    Hyal3 or SPAM1) in this study, human also has hyaluronidase Hyal1    and Hyal2 in cells. According to the reference “Recombinant human    hyaluronidase Hyal-1: Insect cells versus Escherichia coli as    expression system and identification of low molecular weight    inhibitors, Glycobiology, 17(4):444-53, 2007”, it has been found    that recombinant human Hyal1 hyaluronidase produced by insect cell    Drosophila Schneider-2 (DS-2) has the specific activity of 8.6 U/mg    and the best reaction pH value of 3.5˜4.0, but such production    method has a very low output, and it is not applicable for    activation and commercialized production of LMW-HA fragment    injection. In addition, we produce recombinant human Hyal1 and Hyal2    proteins using zooblast (CHO-S cells), pMH3, pMH4 and pMH5    expression vectors rich in GC, and human Hyal1 and Hyal2 cDNA by the    construction manner of the expression system in Embodiment 1. It is    found that the activities of the obtained hyaluronidases Hyal1 and    Hyal2 are significantly lower than PH20 (1×105 U/mg zymoprotein)    produced at present, indicating that the recombinant human Hyal1 and    Hyal2 are not applicable for producing zooblasts.-   2. The output of yeast-expressed neutral human acrosome    hyaluronidase PH20 (or Hyal3 or SPAM1) can reach the level of    commercialization, with the specific activity up to 10,000 IU/mg    zymoprotein, and it is applicable for digestion, activation and    commercialized production of LMW-HA fragment injection. We use the    yeast-expressed neutral human acrosome hyaluronidase PH20, after    enzyme digestion, affinity column chromatography, precipitation,    column chromatography, dialysis, ultrafiltration concentration, etc.    are used in order or respectively for recombinant human    hyaluronidase to remove the contained protein residue of recombinant    human hyaluronidase and produce the HA fragment injection similar to    HA fragment Injection 2 in Embodiment 1 (HA fragments with a    molecular weight distribution of 10 KD-60 KD 100 mg/2˜4 ml, 115˜125    mM of sodium chloride and 1 mM of magnesium ions) not containing    protein residue of recombinant human hyaluronidase, and a clinical    study was carried out for 38 subjects of multiple senility-related    diseases. According to the results of Table 42, although    yeast-expressed neutral human acrosome hyaluronidase PH20 had a    lower specific activity, it had certain influence on the purity of    HA fragment injection, and it could be used for producing HA    fragment injection having a therapeutic effect on human and without    allergic side effect by removing the contained protein residue of    recombinant human hyaluronidase.

TABLE 42 Clinical Effect of HA Fragment Injection 2 Produced by UsingYeast-expressed Neutral Human Acrosome Hyaluronidase PH20 in 38 Subjectsof Multiple Senility-related Diseases Number of samples with Number ofeffective researched Indication treatment samples Facial cosmetology andfacial 38 38 anti-aging Pain relief 12 12 Itching relief 7 7 Enhancingphysical strength 36 38 and energy Reducing subcutaneous fat in 36 38head, face and neck Myofascitis 7 7 Cervical Spondylosis 4 4 itchingSenile Eczema 3 3 Recovering Physical Strength 1 1 and preventing themetastasis of solid tumors after chemotherapy for solid tumorsFunctional recovery after 2 2 cerebral infarction Periodontalinflammation 18 18 Dental ulcer 4 4 Guttural inflammation 14 14Conjunctival inflammation 5 5 Note: Self-control was employed in thisstudy, which refers to the comparison between the subject’s currentstate (after treatment) the state of a period before treatment.

3. We produce the fusion protein of neutral human acrosome hyaluronidasePH20 from the oily seed expression system using the plant of Arabidopsisthaliana through a partner laboratory, and it is found that it can beused for producing the above HA fragment injection having a therapeuticeffect on human and without allergic effect; see Table 43 for thespecific effects. Compared with the high-purity glycosylated recombinanthuman hyaluronidase produced from the zooblast of Chinese hamster ovarycells, this method for producing the fusion protein of hyaluronidasePH20 has low cost, high output and low activity, but the cost isslightly higher for removing the contained fusion protein ofhyaluronidase PH20. In addition, based on our previous researchachievements in LMW-HA for external use, we believe that the plantexpressed hyaluronidase PH20 is applicable for producing LMW-HA aerosol,spray, gel preparations, etc. for external use requiring for low purity.

TABLE 43 Clinical Effect of HA Fragment Injection 2 Produced by UsingPlant-expressed Neutral Human Acrosome Hyaluronidase PH20 in 35 Subjectsof Multiple Senility-related Diseases Number of samples with Number ofeffective researched Indication treatment samples Facial cosmetology andfacial 35 35 anti-aging Pain relief 15 15 Itching relief 7 7 Enhancingphysical strength 30 35 and energy Reducing subcutaneous fat in 30 35head, face and neck myofascitis 7 7 Myofascitis 4 4 Cervical Spondylosis3 3 Itching Senile Eczema 1 1 Recovering Physical Strength 2 2 andpreventing the metastasis of solid tumors after chemotherapy for solidtumors Periodontal inflammation 16 16 Dental ulcer 4 4 Gutturalinflammation 13 13 Conjunctival inflammation 5 5 Note: Self-control wasemployed in this study, which refers to the comparison between thesubject’s current state (after treatment) the state of a period beforetreatment.

-   4. The hyaluronidase extracted from goat or bovine testes is mainly    neutral goat or bovine acrosome hyaluronidase PH20, and the skin    allergy test is required before use for human. Though such    hyaluronidase has low cost, it also has low purity, and the cost is    also high for removing the protein residuals of hyaluronidase.    Compared with high-purity glycosylated recombinant human    hyaluronidase produced from the zooblast of Chinese hamster ovary    cells, it is believed that it is not applicable for producing the HA    fragment injection having a therapeutic effect on human, without    allergic side effect and requiring for high purity.-   5. According to the Patent WO/2014/165713 of CEDARS-SINAI MEDICAL    CENTER, LMW-HA less than 10 KD generated by streptococci    hyaluronidase does not cause inflammatory reactions, but LMW-HA    greater than 10 KD generated by Streptomyces hyaluronidase causes    inflammatory reactions, and these are contradictive to our HA    fragment with a molecular weight distribution of 10 KD-60 KD having    a therapeutic effect; however, it shall be mainly considered here    that the protein residue of bacterial hyaluronidase may induce    allergic reactions for human, and it is not applicable for producing    the HA fragment injection having a therapeutic effect on human,    without allergic side effect and requiring for high purity.-   6. Hyaluronidase bee venom has strong allergen property, and it can    cause allergic reaction for human in addition to promoting the    absorption of bee venom. The hyaluronidase extracted from bee venom    and the microorganism-expressed hyaluronidase in bee venom are both    not applicable for producing the HA fragment injection having a    therapeutic effect on human, without allergic side effect and    requiring for high purity.-   7. Leech hyaluronidase secreted by hematophagous leech can dissolve    skin and help the leech enter human body to suck blood. According to    the references of “Enzymatic production of specifically distributed    hyaluronan oligosaccharides, Carbohydr Polym, 2015, 129:194-200” and    “High-yield novel leech hyaluronidase to expedite the preparation of    specific hyaluronan oligomers (2014), Scientific Reports 4, Article    number 4471”, the yield, digestion activity and cust of the fusion    protein of leech hyaluronidase in yeast expression can meet the    requirement for commercialization and can digest HMW-HA into LMW-HA.    However, the residue of leech hyaluronidase can cause allergic    reactions, and it can be used for producing the HA fragment    injection having a therapeutic effect on human shown in the above    embodiments, without allergic side effect and requiring for high    purity only by complete elimination.

Therefore, by summarizing the above research results, we believe that inthe process of producing LMW-HA fragments in the invention, theapplicable recombinant human hyaluronidase can be produced by CHO cellsor yeast or plant expression. The recombinant human hyaluronidaseobtained by these production methods can meet the requirements forsafety and activity, and besides, the LMW-HA fragment injectionsproduced by these production methods have been validated by clinicalpractices in human, and have shown effective therapeutic effects withoutallergy and side effect.

Finally, it shall be explained that the above are only the optimalembodiments of the invention and are not used for limiting theinvention. Although the invention is explained in details in referenceto the previous embodiments, the technicians of this field can amend thetechnical schemes recorded in the previous embodiments or performequivalent substitution for part of the technical features. Anyamendment, equivalent substitution, improvement, etc. within the energyand principles of the invention shall be within the protective scope ofthe invention.

What is claimed is:
 1. An application of low-molecular-weight hyaluronicacid (LMW-HA) fragments in preparing drugs for treating solid tumors. 2.The application according to claim 1, characterized in that, the solidtumors include lung cancer, pancreatic cancer, oral cancer, meningioma,gastric cancer, and ovarian cancer.
 3. An application of LMW-HAfragments in enhancing or restoring physical strength and energy afterchemotherapy or in preparing drugs for treating side effects afterchemotherapy.
 4. The application according to claim 1, characterized inthat, the LMW-HA fragments are hyaluronic acid (HA) fragments with amolecular weight distribution of 10-60 KD, and the hyaluronic acid (HA)fragments are obtained by directly digesting a high-molecular-weighthyaluronic acid (HMW-HA) raw material using a recombinant humanhyaluronidase PH20.
 5. The application according to claim 4,characterized in that, the recombinant human hyaluronidase PH20 is therecombinant human hyaluronidase produced by zooblasts or yeast or plantexpression, with a purity higher than 98.5%.
 6. The applicationaccording to claim 4, characterized in that, the LMW-HA fragments areprepared into an injection for application, and the injection containsthe effective dose of HA fragments with a molecular weight of 10-60 KD.7. The application according to claim 6, characterized in that, theinjection is injected via abdominal subcutaneous fat layer or atpainful/itchy points or diseased site.
 8. The application according toclaim 6, characterized in that, the dose of injection is 1-2 times perday, and the effective dose of HA fragments is 100-200 mg per injection.9. The application according to claim 6, characterized in that, theinjection is a liquid preparation and is prepared by the followingsteps: A. preparing the HMW-HA raw material into a solution, introducingsodium chloride and magnesium ions, and adding the recombinant humanhyaluronidase PH20 for digestion to obtain a bulk injection containingthe HA fragments with the molecular weight distribution of 10-60 KD; B.inactivating the residual recombinant human hyaluronidase PH20 in theobtained bulk injection; and C. inactivating viruses and removingbacteria through filtration or inactivating bacteria.
 10. Theapplication according to claim 9, characterized in that, the HMW-HA rawmaterial in the Step A are baked at 105° C. for 5-6 h in advance and arethen prepared into the solution.
 11. The application according to claim10, characterized in that, the HMW-HA raw material in the Step A have amolecular weight distribution of 800-1200 KD, 4000-5000 units of therecombinant human hyaluronidase PH20 are added to each 1 g of the HMW-HAraw material correspondingly for digestion, and the enzyme digestionreaction is kept for 5-6 h.
 12. The application according to claim 9,characterized in that, the HMW-HA raw materials in Step A are directlyprepared into solution, the HMW-HA raw materials have a molecular weightof 800-1200 KD, 15000-20000 units of the recombinant human hyaluronidasePH20 are added to each 1 g of the HMW-HA raw material correspondinglyfor digestion, and the enzyme digestion reaction is kept for 5-6 h. 13.The application according to claim 9, characterized in that, theconcentrations of sodium chloride and magnesium ions introduced in StepA in the prepared solution are 80-90 mM and 1 mM, respectively.
 14. Theapplication according to claim 9, characterized in that, the inactivatedrecombinant human hyaluronidase is also removed after Step B.
 15. Theapplication according to claim 9, characterized in that, each 2-4 mL ofthe injection contains 100 mg of HA fragments, and the residualrecombinant human hyaluronidase shall not exceed 20 μg.
 16. Theapplication according to claim 15, characterized in that, the finalconcentration of magnesium ions in the injection is 1 mM, and the finalconcentration of sodium chloride in the injection is 115-125 mM.
 17. Theapplication according to claim 2, characterized in that, the LMW-HAfragments are hyaluronic acid (HA) fragments with a molecular weightdistribution of 10-60 KD, and the hyaluronic acid (HA) fragments areobtained by directly digesting a high-molecular-weight hyaluronic acid(HMW-HA) raw material using a recombinant human hyaluronidase PH20. 18.The application according to claim 17, characterized in that, therecombinant human hyaluronidase PH20 is the recombinant humanhyaluronidase produced by zooblasts or yeast or plant expression, with apurity higher than 98.5%.
 19. The application according to claim 17,characterized in that, the LMW-HA fragments are prepared into aninjection for application, and the injection contains the effective doseof HA fragments with a molecular weight of 10-60 KD.
 20. The applicationaccording to claim 3, characterized in that, the LMW-HA fragments arehyaluronic acid (HA) fragments with a molecular weight distribution of10-60 KD, and the hyaluronic acid (HA) fragments are obtained bydirectly digesting a high-molecular-weight hyaluronic acid (HMW-HA) rawmaterial using a recombinant human hyaluronidase PH20.